Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites

ABSTRACT

The present invention provides compositions and methods for recombinational cloning. The compositions include vectors having multiple recombination sites and/or multiple topoisomerase recognition sites. The methods permit the simultaneous cloning of two or more different nucleic acid molecules. In some embodiments the molecules are fused together while in other embodiments the molecules are inserted into distinct sites in a vector. The invention also generally provides for linking or joining through recombination a number of molecules and/or compounds (e.g., chemical compounds, drugs, proteins or peptides, lipids, nucleic acids, carbohydrates, etc.) which may be the same or different. The invention also provides host cells comprising nucleic acid molecules of the invention or prepared according to the methods of the invention, and also provides kits comprising the compositions, host cells and nucleic acid molecules of the invention, which may be used to synthesize nucleic acid molecules according to the methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/792,035 filed Mar. 4, 2004, which is a continuation of U.S.application Ser. No. 10/454,793, filed Jun. 5, 2003, which claims thebenefit of the filing date of U.S. provisional patent application No.60/385,613, filed Jun. 5, 2002. This application U.S. application Ser.No. 10/454,793 also is a continuation-in-part of U.S. application Ser.No. 10/005,876, filed Dec. 7, 2001, which claims the benefit of thefiling dates of U.S. provisional application No. 60/333,124, filed Nov.27, 2001, and 60/318,902, filed Sep. 14, 2001. This application also isa continuation-in-part of U.S. application Ser. No. 10/014,128, filedDec. 7, 2001, and of U.S. application Ser. No. 09/732,914, filed Dec.11, 2000. The disclosures of all of the above-referenced applicationsare specifically incorporated herein by reference in their entireties.

U.S. patent application Ser. No. 09/935,280, filed Aug. 21, 2001, andU.S. provisional patent application No. 60/326,092, filed Sep. 28, 2001,60/291,972, filed May 21, 2001, 60/254,510, filed Dec. 8, 2000, and60/226,563, filed Aug. 21, 2000, also are specifically incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fields of biotechnology andmolecular biology. In particular, the present invention relates tojoining multiple nucleic acid molecules containing one or morerecombination sites and/or one or more topoisomerase recognition sites.The present invention also relates to cloning such joined nucleic acidmolecules using recombinational cloning methods such as those employingtopoisomerase and/or recombination proteins. The invention also relatesto joining multiple peptides, and combinations of peptides and nucleicacid molecules through the use of recombination sites and/ortopoisomerase recognition sites. Other molecules and compounds orcombinations of molecules and compounds may also be joined throughrecombination sites and/or topoisomerase recognition sites according tothe invention. Such peptides, nucleic acids and other molecules and/orcompounds (or combinations thereof) may also be joined or bound throughrecombination reactions and/or through topoisomerase joining reactionsto one or a number of supports or structures in accordance with theinvention.

2. Related Art

Site-Specific Recombinases

Site-specific recombinases are proteins that are present in manyorganisms (e.g. viruses and bacteria) and have been characterized ashaving both endonuclease and ligase properties. These recombinases(along with associated proteins in some cases) recognize specificsequences of bases in a nucleic acid molecule and exchange the nucleicacid segments flanking those sequences. The recombinases and associatedproteins are collectively referred to as “recombination proteins” (see,e.g., Landy, A., Current Opinion in Biotechnology 3:699-707 (1993)).

Numerous recombination systems from various organisms have beendescribed. See, e.g., Hoess, et al., Nucleic Acids Research 14(6):2287(1986); Abremski, et al., J. Biol. Chem. 261(1):391 (1986); Campbell, J.Bacteriol. 174(23):7495 (1992); Qian, et al., J. Biol. Chem.267(11):7794 (1992); Araki, et al., J. Mol. Biol. 225(1):25 (1992);Maeser and Kahnmann, Mol. Gen. Genet. 230:170-176) (1991); Esposito, etal., Nucl. Acids Res. 25(18):3605 (1997). Many of these belong to theintegrase family of recombinases (Argos, et al., EMBO J. 5:433-440(1986); Voziyanov, et al., Nucl. Acids Res. 27:930 (1999)). Perhaps thebest studied of these are the Integrase/att system from bacteriophage(Landy, A. Current Opinions in Genetics and Devel. 3:699-707 (1993)),the Cre/loxP system from bacteriophage P1 (Hoess and Abremski (1990) InNucleic Acids and Molecular Biology, vol. 4. Eds.: Eckstein and Lilley,Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the FLP/FRT systemfrom the Saccharomyces cerevisiae 2μ circle plasmid (Broach, et al.,Cell 29:227-234 (1982)).

Recombination Sites

Whether the reactions discussed above are termed recombination,transposition or integration and are catalyzed by a recombinase,transposase or integrase, they share the key feature of specificrecognition sequences, often termed “recombination sites,” on thenucleic acid molecules participating in the reactions. Theserecombination sites are sections or segments of nucleic acid on theparticipating nucleic acid molecules that are recognized and bound bythe recombination proteins during the initial stages of integration orrecombination. For example, the recombination site for Cre recombinaseis loxP which is a 34 base pair sequence comprised of two 13 base pairinverted repeats (serving as the recombinase binding sites) flanking an8 base pair core sequence. See FIG. 1 of Sauer, B., Curr. Opin. Biotech.5:521-527 (1994). Other examples of recognition sequences include theattB, attP, attL, and attR sequences which are recognized by therecombination protein (Int. attB is an approximately 25 base pairsequence containing two 9 base pair core-type Int binding sites and a 7base pair overlap region, while attP is an approximately 240 base pairsequence containing core-type Int binding sites and arm-type Int bindingsites as well as sites for auxiliary proteins integration host factor(IHF), FIS and excisionase (Xis). See Landy, Curr. Opin. Biotech.3:699-707 (1993).

Conventional Nucleic Acid Cloning

The cloning of nucleic acid segments currently occurs as a daily routinein many research labs and as a prerequisite step in many geneticanalyses. The purpose of these clonings is various, however, two generalpurposes can be considered: (1) the initial cloning of nucleic acid fromlarge DNA or RNA segments (chromosomes, YACs, PCR fragments, mRNA,etc.), done in a relative handful of known vectors such as pUC, pGem,pBlueScript, and (2) the subcloning of these nucleic acid segments intospecialized vectors for functional analysis. A great deal of time andeffort is expended both in the transfer of nucleic acid segments fromthe initial cloning vectors to the more specialized vectors. Thistransfer is called subcloning.

The basic methods for cloning have been known for many years and havechanged little during that time. A typical cloning protocol is asfollows:

(1) digest the nucleic acid of interest with one or two restrictionenzymes;

(2) gel purify the nucleic acid segment of interest when known;

(3) prepare the vector by cutting with appropriate restriction enzymes,treating with alkaline phosphatase, gel purify etc., as appropriate;

(4) ligate the nucleic acid segment to the vector, with appropriatecontrols to eliminate background of uncut and self-ligated vector;

(5) introduce the resulting vector into an E. coli host cell;

(6) pick selected colonies and grow small cultures overnight;

(7) make nucleic acid minipreps; and

(8) analyze the isolated plasmid on agarose gels (often after diagnosticrestriction enzyme digestions) or by PCR.

The specialized vectors used for subcloning nucleic acid segments arefunctionally diverse. These include but are not limited to: vectors forexpressing nucleic acid molecules in various organisms; for regulatingnucleic acid molecule expression; for providing tags to aid in proteinpurification or to allow tracking of proteins in cells; for modifyingthe cloned nucleic acid segment (e.g., generating deletions); for thesynthesis of probes (e.g., riboprobes); for the preparation of templatesfor nucleic acid sequencing; for the identification of protein codingregions; for the fusion of various protein-coding regions; to providelarge amounts of the nucleic acid of interest, etc. It is common that aparticular investigation will involve subcloning the nucleic acidsegment of interest into several different specialized vectors.

As known in the art, simple subclonings can be done in one day (e.g.,the nucleic acid segment is not large and the restriction sites arecompatible with those of the subcloning vector). However, many othersubclonings can take several weeks, especially those involving unknownsequences, long fragments, toxic genes, unsuitable placement ofrestriction sites, high backgrounds, impure enzymes, etc. One of themost tedious and time consuming type of subcloning involves thesequential addition of several nucleic acid segments to a vector inorder to construct a desired clone. One example of this type of cloningis in the construction of gene targeting vectors. Gene targeting vectorstypically include two nucleic acid segments, each identical to a portionof the target gene, flanking a selectable marker. In order to constructsuch a vector, it may be necessary to clone each segment sequentially,i.e., first one gene fragment is inserted into the vector, then theselectable marker and then the second fragment of the target gene. Thismay require a number of digestion, purification, ligation and isolationsteps for each fragment cloned. Subcloning nucleic acid fragments isthus often viewed as a chore to be done as few times as possible.

Several methods for facilitating the cloning of nucleic acid segmentshave been described, e.g., as in the following references.

Ferguson, J., et al., Gene 16:191 (1981), disclose a family of vectorsfor subcloning fragments of yeast nucleic acids. The vectors encodekanamycin resistance. Clones of longer yeast nucleic acid segments canbe partially digested and ligated into the subcloning vectors. If theoriginal cloning vector conveys resistance to ampicillin, nopurification is necessary prior to transformation, since the selectionwill be for kanamycin.

Hashimoto-Gotoh, T., et al., Gene 41:125 (1986), disclose a subcloningvector with unique cloning sites within a streptomycin sensitivity gene;in a streptomycin-resistant host, only plasmids with inserts ordeletions in the dominant sensitivity gene will survive streptomycinselection.

Notwithstanding the improvements provided by these methods, traditionalsubclonings using restriction and ligase enzymes are time consuming andrelatively unreliable. Considerable labor is expended, and if two ormore days later the desired subclone can not be found among thecandidate plasmids, the entire process must then be repeated withalternative conditions attempted.

Recombinational Cloning

Cloning systems that utilize recombination at defined recombinationsites have been previously described in U.S. Pat. Nos. 5,888,732,6,143,557, 6,171,861, 6,270,969, and 6,277,608 which are specificallyincorporated herein by reference. In brief, the Gateway™ Cloning System,described in this application and the applications referred to in therelated applications section, utilizes vectors that contain at least oneand preferably at least two different site-specific recombination sitesbased on the bacteriophage lambda system (e.g., att1 and att2) that aremutated from the wild type (att0) sites. Each mutated site has a uniquespecificity for its cognate partner att site of the same type (forexample attB1 with attP1, or attL1 with attR1) and will not cross-reactwith recombination sites of the other mutant type or with the wild-typeatt0 site. Nucleic acid fragments flanked by recombination sites arecloned and subcloned using the Gateway™ system by replacing a selectablemarker (for example, ccdB) flanked by att sites on the recipient plasmidmolecule, sometimes termed the Destination Vector. Desired clones arethen selected by transformation of a ccdB sensitive host strain andpositive selection for a marker on the recipient molecule. Similarstrategies for negative selection (e.g., use of toxic genes) can be usedin other organisms such as thymidine kinase (TK) in mammals and insects.

Mutating specific residues in the core region of the att site cangenerate a large number of different att sites. As with the att1 andatt2 sites utilized in Gateway™, each additional mutation potentiallycreates a novel alt site with unique specificity that will recombineonly with its cognate partner aut site bearing the same mutation andwill not cross-react with any other mutant or wild-type att site. Novelmutated alt sites (e.g., attB 1-10, attP 1-10, attR 1-10 and attL 1-10)are described in commonly owned U.S. application Ser. No. 09/517,466,filed Mar. 2, 2000, which is specifically incorporated herein byreference. Other recombination sites having unique specificity (i.e., afirst site will recombine with its corresponding site and will notrecombine or not substantially recombine with a second site having adifferent specificity) may be used to practice the present invention.Examples of suitable recombination sites include, but are not limitedto, loxP sites and derivatives such as loxP511 (see U.S. Pat. No.5,851,808), frt sites and derivatives, dif sites and derivatives, psisites and derivatives and cer sites and derivatives. The presentinvention provides novel methods using such recombination sites to joinor link multiple nucleic acid molecules or segments and morespecifically to clone such multiple segments into one or more vectorscontaining one or more recombination sites (such as any Gateway™ Vectorincluding Destination Vectors).

SUMMARY OF THE INVENTION

The invention relates; in part, to nucleic acid molecules which compriseone or more (e.g., one, two, three, four, five, etc.) recombinationsites (e.g., one or more alt sites, one or more lox sites, etc.) and/orone or more (e.g., one, two, three, four, five, etc.) topoisomeraserecognition sites (e.g., one or more recognition sites for a type IAtopoisomerase, a type IB topoisomerase, a type II topoisomerase, etc.),as well as nucleic acid molecules which have undergone cleavage with atopoisomerase (e.g., a site specific topoisomerase). The invention alsorelates to nucleic acid molecules which comprise one or morerecombination sites and/or one or more topoisomerases. The inventionmore specifically relates to combining or joining at least a firstnucleic acid molecule which comprises at least a first nucleic acidmolecule which comprises at least one recombination site and at least asecond nucleic acid molecule which comprises at least one topoisomeraserecognition site and/or at least one topoisomerase. Upon joining theseat least first and second molecules, at least a third (or chimeric)molecule may be produced which comprises (1) at least one recombinationsite and (2) at least one topoisomerase recognition site and/or at leastone topoisomerase. These nucleic acid molecules may be linear or closedcircular (e.g., relaxed, supercoiled, etc.). Such recombination sites,topoisomerase recognition sites and topoisomerase can be located at anyposition on any number of nucleic acid molecules of the invention,including at or near the termini of the nucleic acid molecules and/orwithin the nucleic acid molecules. Moreover, any combination of the sameor different recombination sites, topoisomerase recognition sites and/ortopoisomerases may be used in accordance with the invention.

The invention includes, in part, nucleic acid molecules and compositionscomprising nucleic acid molecules (e.g., reaction mixtures), wherein thenucleic acid molecules comprise (1) at least one (e.g., one, two, three,four, five, six, seven eight, etc.) recombination site and (2) at leastone (e.g., one, two, three, four, five, six, seven eight, etc.)topoisomerase (e.g., a covalently linked topoisomerase) or at least one(e.g., one, two, three, four, five, six, seven eight, etc.)toposiomerase recognition site. In particular embodiments, thetopoisomerases or toposiomerase recognition sites, as well as therecombination sites, of the nucleic acid molecules referred to above canbe either internal or at or near one or both termini. For example, oneor more (e.g., one, two, three, four, five, six, seven eight, etc.) ofthe at least one topoisomerase or the at least one topoisomeraserecognition site, as well as one or more of the at least onerecombination site, can be located at or near a 5′ terminus, at or neara 3′ terminus, at or near both 5′ termini, at or near both 3′ termini,at or near a 5′ terminus and a 3′ terminus, at or near a 5′ terminus andboth 3′ termini, or at or near a 3′ terminus and both 5′ termini. Theinvention further provides methods for preparing and using nucleic acidmolecules and compositions of the invention.

In specific aspects, the invention provides nucleic acid molecules (1)to which topoisomerases of various types (e.g., a type IA toposiomerase,a type IB toposiomerase, a type II topoisomerase, etc.) are attached(e.g., covalently bound) and/or (2) which contain two or moretopoisomerase recognition sites which are recognized by various types oftopoisomerases, as well as methods for preparing and using compositionscomprising such nucleic acid molecules. In many embodiments, thesenucleic acid molecules will further comprise one or more (e.g., one,two, three, four, five, six, seven eight, etc.) recombination site.

The invention further provides methods for joining two or more nucleicacid segments, wherein at least one of the nucleic acid segmentscontains at least one toposiomerase or topoisomerase recognition siteand/or one or more recombination sites. Further, when nucleic acidsegments used in methods of the invention contain more than one (e.g.,two, three, four, five, six, seven eight, etc.) toposiomerase, either onthe same or different nucleic acid segments, these toposiomerase may beof the same type or of different types. Similarly, when nucleic acidsegments used in methods of the invention contain more than onetoposiomerase recognition site, either on the same or different nucleicacid segments, these toposiomerase recognition sites may be recognizedby topoisomerases of the same type or of different types. Additionally,when nucleic acid segments used in methods of the invention contain oneor more recombination sites, these recombination sites may be able torecombine with one or more recombination sites on the same or differentnucleic acid segments. Thus, the invention provides methods for joiningnucleic acid segments using methods employing any one toposiomerase ortopoisomerase recognition site. The invention provides further methodsfor joining nucleic acid segments using methods employing (1) anycombination of topoisomerases or topoisomerase recognition sites and/or(2) any combination of recombination sites. The invention also providesnucleic acid molecules produced by the methods described above, as wellas uses of these molecules and compositions comprising these molecules.

In general, the invention provides, in part, methods for joining anynumber of nucleic acid segments (e.g., two, three, four, five, six,seven, eight, nine, ten, etc.) which contain different functional orstructural elements. The invention thus provides, in part, methods forbringing together any number of nucleic acid segments (e.g., two, three,four, five, six, seven, eight, nine, ten, etc.) which confer differentproperties upon a nucleic acid molecule product. In many instances,methods of the invention will result in the formation of nucleic acidmolecules wherein there is operable interaction between propertiesand/or elements of individual nucleic acid segments which are joined(e.g., operable interaction/linkage between an expression controlsequence and an open reading frame). Examples of (1) functional andstructural elements and (2) properties which may be conferred uponproduct molecules include, but are not limited to, multiple cloningsites (e.g., nucleic acid regions which contain at least two restrictionendonuclease cleavage sites), packaging signals (e.g., adenoviralpackaging signals, alphaviral packaging signals, etc.), restrictionendonuclease cleavage sites, open reading frames (e.g., intein codingsequence, affinity purification tag coding sequences, etc.), expressioncontrol sequences (e.g., promoters, operators, etc.), etc. Additionalelements and properties which can be conferred by nucleic acid segmentsupon a product nucleic acid molecule are described elsewhere herein. Theinvention also provides nucleic acid molecules produced by the methodsdescribed above, as well as uses of these molecules and compositionscomprising these molecules.

The invention further includes, in part, methods for joining two or more(e.g., 2, 3, 4, 5, 6, 7, 8, etc.) nucleic acid segments, wherein atleast one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) of the nucleic acidsegments comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.)topoisomerases and/or one or more topoisomerase recognition sites and atleast one of the nucleic acid segments comprises one or morerecombination sites. In particular embodiments, the invention providesmethods for joining at least two (e.g., 2, 3, 4, 5, 6, 7, 8, etc.)nucleic acid molecules (e.g., methods employing recombination and/ormediated by one or more topoisomerases), wherein one of the nucleic acidsegments comprises one or more topoisomerases or topoisomeraserecognition sites but does not contain a recombination site and theother nucleic acid segments comprises one or more recombination site butdoes not contain a topoisomerase or topoisomerase recognition site.Thus, methods of the invention can be used to prepare joined or chimericnucleic acid molecules by the joining of nucleic acid segments, whereinthe product nucleic acid molecules comprise (1) one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, etc.) topoisomerases and/or one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, etc.) topoisomerase recognition sites and (2) one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) recombination sites. Theinvention further provides nucleic acid molecules prepared by suchmethods, compositions comprising such nucleic acid molecules, andmethods for using such nucleic acid molecules.

The invention also provides compositions comprising one or more nucleicacid segments and/or nucleic acid molecules described herein. Suchcompositions may comprise one or a number of other components selectedfrom the group consisting of one or more other nucleic acid molecules(which may comprise recombination sites, topoisomerase recognitionsites, topoisomerases, etc.), one or more nucleotides, one or morepolymerases, one or more reverse transcriptases, one or morerecombination proteins, one or more topoisomerases, one or more buffersand/or salts, one or more solid supports, one or more polyamines, one ormore vectors, one or more restriction enzymes and the like. For example,compositions of the invention include, but are not limited to, mixtures(e.g., reaction mixtures) comprising a nucleic acid segment whichcomprises at least one topoisomerase recognition site and at least onetopoisomerase which recognizes at least one of the at least onetopoisomerase recognition sites of the nucleic acid segment.Compositions of the invention further include at least one nucleic acidsegment comprising (1) at least one topoisomerase recognition site or atleast one nucleic acid segment to which at least one topoisomerase isattached (e.g., covalently bound) and (2) one or more additionalcomponents. Examples of such additional components include, but are notlimited to, topoisomerases; additional nucleic acid segments, which mayor may not comprise one or more topoisomerases or topoisomeraserecognition sites; buffers; salts; polyamines (e.g., spermine,spermidine, etc.); water; etc. Nucleic acid segments present incompositions of the invention may further comprise one or morerecombination sites and/or one or more recombinase.

Nucleic acid molecules or segments produced by or used in conjunctionwith the methods of the invention, as well as nucleic acid molecules orsegments thereof of the invention, include those molecules or segmentsspecifically described herein as well as those molecules or segmentsthat have substantial sequence identity to those molecules or segmentsspecifically described herein. By a molecule or segment having“substantial sequence identity” to a given molecule or segment is meantthat the molecule or segment is at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99%, identical to the given (or “reference”)molecule or segment. By a nucleic acid molecule or segment having anucleotide sequence at least, for example, 65% “identical” to areference nucleic acid molecule or segment is intended that thenucleotide sequence of the nucleic acid molecule or segment is identicalto that of the reference sequence except that the nucleic acid moleculeor segment may include up to 35 point mutations per each 100 nucleotidesof the reference nucleotide sequence. In other words, to obtain apolynucleotide having a nucleotide sequence at least 65% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 35% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5′or 3′ terminal positions (or both) of the reference nucleotide sequence,or anywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule orsegment is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to a given reference molecule or segment can bedetermined conventionally using known computer programs such as FASTA(Heidelberg, Germany), BLAST (Washington, D.C.) or BESTFIT (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711), whichemploys a local homology algorithm (Smith and Waterman, Advances inApplied Mathematics 2: 482-489 (1981)) to find the best segment ofhomology between two sequences. When using FASTA, BLAST, BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for instance, 65% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 35% ofthe total number of nucleotides in the reference sequence are allowed.

Often, nucleic acid molecules which have undergone cleavage with atopoisomerase (e.g., a site specific topoisomerase) will further have atopoisomerase molecule covalently bound to a phosphate group of thenucleic acid molecules. The invention further includes methods forpreparing nucleic acid molecules described above and elsewhere herein,as well as recombinant methods for using such molecules.

In particular embodiments, nucleic acid molecules of the invention willbe vectors. In additional embodiments, the invention includes host cellswhich contain nucleic acid molecules of the invention, as well asmethods for making and using such host cells, for example, to produceexpression products (e.g., proteins, polypeptides, antigens, antigenicdeterminants, epitopes, and the like, or fragments thereof).

In specific embodiments, nucleic acid molecules of the inventioncomprise two or more recombination sites with one or more (e.g., one,two, three, four, five, etc.) topoisomerase recognition site locatedbetween the recombination sites. In additional specific embodiments,nucleic acid molecules of the invention may comprise two or moretopoisomerase recognition sites with one or more (e.g., one, two, three,four, five, etc.) recombination sites located between the two or moretopoisomerase recognition sites.

In additional specific embodiments, nucleic acid molecules of theinvention comprise two recombination sites with two topoisomeraserecognition sites located between the two recombination sites. Thus, ifsuch molecules are linearized by cleavage between the topoisomeraserecognition sites, the topoisomerase recognition sites in the resultinglinear molecule will be located distal (i.e., closer to the two ends ofthe linear molecule) to the recombination sites. The invention thusprovides linear nucleic acid molecules which contain one or morerecombination sites and one or more topoisomerase recognition sites. Inparticular embodiments, the one or more topoisomerase recognition sitesare located distal to the one or more recombination sites. Examples ofsuch molecules are set out below in Example 8.

The positioning of recombination sites and topoisomerase recognitionsites of a first nucleic acid molecule can be such that topoisomerasemediated linkage of this molecule to a second nucleic acid moleculeresults in the second nucleic acid molecule being positioned between thetwo or more recombination sites. As an example, a linear first nucleicacid molecule may contain one recombination site at or near each end andmay further comprise a topoisomerase recognition site located distal toone of the two recombination sites. In such a case, incubation of thelinear first nucleic acid molecule with a topoisomerase can be designedto result in the covalent linkage of the topoisomerase to the firstnucleic acid molecule, wherein the topoisomerase is positioned at ornear the end of the first nucleic acid molecule and distal to theadjacent/nearest recombination site. This end of the first nucleic acidmolecule may be blunt or may have either a 5′ or 3′ overhang. Whenincubated with a suitable second nucleic acid molecule (e.g., a moleculewith sequence complementarity to at least one strand of thetopoisomerase modified end of the first nucleic acid molecule), one orboth strands of one end of the second nucleic acid molecule can becovalently joined to one or both strands of one end of the first nucleicacid molecule. Further, if a circular nucleic acid molecule is desired,then the second end of the second nucleic acid molecule can be joined tothe second end of the first nucleic acid molecule by a topoisomerase, aligase or other method. The result of the process described above is thegeneration of a nucleic acid molecule which contains a nucleic acidinsert positioned between two recombination sites. Specific examples ofrelated processes are set out below in Example 8. Methods for covalentlylinking nucleic acid molecules using topoisomerase are described in moredetail elsewhere herein.

Once a nucleic acid insert has been positioned between one or morerecombination sites, this insert, as well as adjacent nucleic acid, maybe transferred to other nucleic acid molecules by recombinationalcloning. The invention thus also provides methods for generating thenucleic acid molecules described above and elsewhere herein.

The distance, in terms of the number of nucleotides, betweenrecombination sites and topoisomerase recognition sites which reside ina nucleic acid molecule of the invention will vary with the particularapplication for which the molecule is to be used, but can be zero, one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, twenty, twenty-five, thirty,forty, fifty, sixty, eighty, one hundred, one hundred fifty, twohundred, three hundred, five hundred, seven hundred, nine hundred, onethousand, etc., or more, nucleotides. Further, the distance, in terms ofthe number of nucleotides, between recombination sites and topoisomeraserecognition sites which reside in a nucleic acid molecule of theinvention may fall within the following ranges: 0-10 nucleotides, 10-30nucleotides, 20-50 nucleotides, 40-80 nucleotides, 70-100 nucleotides,90-200 nucleotides, 120-400 nucleotides, 200-400 nucleotides, 200-1000nucleotides, 200-2,000 nucleotides, etc.

The present invention also generally provides materials and methods forjoining or combining two or more (e.g., three or more, four or more,five or more etc.) segments or molecules of nucleic acid of theinvention. In one aspect, for such molecules to be combined, at leastone of the segments or molecules may comprise at least one recombinationsite and at least one of the segments or molecules may comprise at leastone topoisomerase recognition site. Such methods for joining multiplenucleic acid molecules according to the invention may be conducted invivo or in vitro. Accordingly, the invention relates to methods tocreate novel or unique combinations of sequences and to the sequencescreated by such methods. The nucleic acid molecules created by themethods of the invention may be used for any purpose known to thoseskilled in the art. In one aspect, at least one (and often two or more)of the nucleic acid molecules or segments to be joined by the methods ofthe invention comprise at least one, and preferably at least two,recombination sites, although each molecule may comprise multiplerecombination sites (e.g., three or more, four or more, five or more,etc.). In another aspect, the nucleic acid molecules may comprise atleast one topoisomerase recognition site and/or at least onetopoisomerase. In yet another aspect, the molecules may comprise (1) atleast one recombination site and (2) at least one topoisomeraserecognition site and/or at least one topoisomerase. Such recombinationsites and topoisomerase recognition sites (which may be the same ordifferent) may be located at various positions in each nucleic acidmolecule or segment and the nucleic acid used in the invention may havevarious sizes and be in different forms including circular, supercoiled,linear, and the like. The nucleic acid molecules used in the inventionmay also comprise one or more vectors or one or more sequences allowingthe molecule to function as a vector in a host cell (such as an originof replication). In one aspect, nucleic acid molecules or segments foruse in the invention are linear molecules having at least onerecombination site at or near at least one termini of the molecule andpreferably comprise at least one recombination site at or near bothtermini of the molecule. In another aspect, when multiple recombinationsites are located on a nucleic acid molecule of interest, such sites donot substantially recombine or do not recombine with each other on thatmolecule. In this embodiment, the corresponding binding partnerrecombination sites preferably are located on one or more other nucleicacid molecules to be linked or joined by the methods of the invention.For instance, a first nucleic acid molecule used in the invention maycomprise at least a first and second recombination site and a secondnucleic acid molecule may comprise at least a third and fourthrecombination site, wherein the first and second sites do not recombinewith each other and the third and fourth sites do not recombine witheach other, although the first and third and/or the second and fourthsites may recombine.

The nucleic acid molecules to be joined by the methods of the invention(e.g., the “starting molecules”) may be used to produce one or morehybrid molecules containing all or a portion of the starting molecules(e.g., the “product nucleic acid molecules”). The starting molecules canbe any nucleic acid molecule derived from any source or produced by anymethod. Such molecules may be derived from natural sources (such ascells, tissue, and organs from any animal or non-animal source) or maybe non-natural (e.g., derivative nucleic acids) or syntheticallyderived. The segments or molecules for use in the invention may beproduced by any means known to those skilled in the art including, butnot limited to, amplification such as by PCR, isolation from naturalsources, chemical synthesis, shearing or restriction digest of largernucleic acid molecules (such as genomic or cDNA), transcription, reversetranscription and the like, and recombination sites and/or topoisomeraserecognition sites and/or topoisomerases may be added to such moleculesby any means known to those skilled in the art including ligation ofadapters containing recombination sites and/or topoisomerase recognitionsites and/or topoisomerases, amplification or nucleic acid synthesisusing primers containing recombination sites and/or topoisomeraserecognition sites and/or topoisomerases, insertion or integration ofnucleic acid molecules (e.g., transponsons or integration sequences)containing recombination sites and/or topoisomerase recognition sitesand/or topoisomerases, etc. In one aspect, the nucleic acid moleculesused in the invention are populations of molecules such as nucleic acidlibraries or cDNA libraries.

Once nucleic acid molecules are joined by recombination using methodssuch as those described herein, these nucleic acid molecules may then bejoined to other nucleic acid molecules using topoisomerase-mediatedjoining methods and/or recombination-mediated joining methods alsodescribed herein.

Recombination sites for use in the invention may be any recognitionsequence on a nucleic acid molecule which participates in arecombination reaction catalyzed or facilitated by recombinationproteins. In those embodiments of the present invention utilizing morethan one recombination site, such recombination sites may be the same ordifferent and may recombine with each other or may not recombine or notsubstantially recombine with each other. Recombination sitescontemplated by the invention also include mutants, derivatives orvariants of wild-type or naturally occurring recombination sites.Preferred recombination site modifications include those that enhancerecombination, such enhancement selected from the group consisting ofsubstantially (i) favoring integrative recombination; (ii) favoringexcisive recombination; (iii) relieving the requirement for hostfactors; (iv) increasing the efficiency of co-integrate or productformation; and (v) increasing the specificity of co-integrate or productformation. Preferred modifications include those that enhancerecombination specificity, remove one or more stop codons, and/or avoidhair-pin formation. Desired modifications can also be made to therecombination sites to include desired amino acid changes to thetranscription or translation product (e.g., mRNA or protein) whentranslation or transcription occurs across the modified recombinationsite. Recombination sites that may be used in accordance with theinvention include att sites, frt sites, dif sites, psi sites, cer sites,and lox sites or mutants, derivatives and variants thereof (orcombinations thereof). Recombination sites contemplated by the inventionalso include portions of such recombination sites.

Each starting nucleic acid molecule may comprise, in addition to one ormore recombination sites and/or one or more topoisomerase recognitionsites and/or one or more topoisomerases, a variety of sequences (orcombinations thereof) including, but not limited to sequences suitablefor use as primer sites (e.g., sequences which a primer such as asequencing primer or amplification primer may hybridize to initiatenucleic acid synthesis, amplification or sequencing), transcription ortranslation signals or regulatory sequences such as promoters and/oroperators, ribosomal binding sites, topoisomerase recognition sequences(or sites), Kozak sequences, and start codons, transcription and/ortranslation termination signals such as stop codons (which may beoptimally suppressed by one or more suppressor tRNA molecules), tRNAs(e.g., suppressor tRNAs), origins of replication, selectable markers,and genes or portions of genes which may be used to create proteinfusion (e.g., N-terminal or carboxy terminal) such as GST, GUS, GFP,open reading frame (orf) sequences, and any other sequence of interestwhich may be desired or used in various molecular biology techniquesincluding sequences for use in homologous recombination (e.g., genetargeting).

The present invention also relates to methods of generating a covalentlylinked recombinant nucleic acid molecule by contacting two or more(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc) nucleic acid molecules (whichmay be alternatively and equivalently referred to herein as “nucleotidesequences”), e.g., double-stranded (“ds”) or single-stranded (“ss”)nucleic acid molecules, with at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, etc.) topoisomerase. As will be understood by the ordinarilyskilled artisan, any and all of the nucleic acid molecules or nucleotidesequences referred to herein, for example those used in or generated bythe methods, compositions and kits disclosed herein, may be ss or dsnucleic acid molecules or nucleotide sequences, whether or not themolecules or sequences are specifically referred to herein as being ssand/or ds.

In one such aspect, the methods of the invention allow joining of suchnucleic acid sequences in a desired orientation and/or order, which, ifdesired, can be further manipulated or used in a variety of assays orprocedures, including, for example, for a transcription or transfectionprocedure, which can be performed in vitro or in vivo, a translationreaction or other protein expression procedure, recombination reactions,and the like. In another aspect, three or more, four or more, five ormore, etc., or a population or library of the same or different nucleicacid sequences can be linked according to a method of the invention. Instill another aspect, the methods of the invention can be used to linkeach end of a single nucleic acid molecule to form a covalently closedcircular or supercoiled molecule.

The nucleic acid sequences to be linked can be derived from any source,and can be naturally occurring and chemically or recombinantlysynthesized nucleic acid molecules such as cDNA, genomic DNA, vectors,oligonucleotides, and the like. Furthermore, the nucleic acid sequencescan, but need not, contain one or more functional sequences such as generegulatory elements, origins of replication, splice sites,polyadenylation sites, open reading frames, which can encode, forexample, tag sequences, detectable or selectable markers, celllocalization domains, or other peptide or polypeptide, and the like. Assuch, the invention allows any number of nucleic acid sequences, whichcan be the same or different, to be linked, including, if desired, in apredetermined order or orientation or both.

The nucleic acid molecules (e.g., ds or ss nucleic acid molecules) to belinked can be in any form, for example, single-stranded ordouble-stranded, linear, circular, or supercoiled, and arecharacterized, in part, in that each nucleic acid molecule to be linkedis a substrate for a topoisomerase or can be modified to be such asubstrate. The topoisomerase can be any topoisomerase that cancovalently link at least one strand of a nucleic acid molecule to atleast one strand of another nucleic acid molecule, preferably through aphosphodiester bond. The topoisomerase can be a site specifictopoisomerase or can have relaxed specificity, and preferably forms astable complex (e.g., a covalent complex) with one strand of the nucleicacid molecule at or near the site at which cleavage is effected.

A method of the invention generally is performed by contactingtopoisomerase and the nucleic acid molecules (e.g., ds or ss nucleicacid molecules) to be joined under conditions such that both strands ofan end of one nucleic acid molecule are ligated to both strands of anend of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) othernucleic acid molecule. As such, a method of the invention generates acovalently linked recombinant nucleic acid molecule (which may be eithersingle-stranded or double-stranded), which does not contain a nick atthe site or sites at which the substrate nucleic acid molecules areligated. The present invention also provides recombinant nucleic acidmolecules prepared by such a method. In certain such aspects of theinvention, such recombinant nucleic acid molecules will further compriseone or more recombination sites.

A method of the invention can be performed using various combinations ofcomponents. For example, the method can be performed by contacting twoor more substrate nucleic acid molecules (e.g., ss nucleic acidmolecules or ds nucleic acid molecules) to be covalently linked and atleast one topoisomerase, wherein the topoisomerase cleaves one or bothstrands of the nucleic acid molecules and forms a stable complex with anucleotide at a terminus of the cleavage site. The topoisomerase-chargedends or topoisomerase-charged nucleic acid molecules are then contactedwith each other such that each strand of the substrate nucleic acidmolecules are linked, thereby generating one or more covalently linkedrecombinant nucleic molecules. Preferably, the topoisomerase mediatesthe formation of phosphodiester bond at each linkage site. The methodalso can be performed by contacting two or more topoisomerase-chargednucleic acid molecules, either alone, or in the presence of excesstopoisomerase, or by contacting one or more topoisomerase-chargednucleic acid molecules (which may be ss or ds) with one or more nucleicacid molecules (which may also be ss or ds) that contain a topoisomerasecleavage site, and a topoisomerase. The present invention also providesrecombinant nucleic acid molecules prepared by such a method. In certainsuch aspects of the invention, such recombinant nucleic acid moleculeswill further comprise one or more recombination sites. In variousembodiments, the topoisomerase can have a relatively relaxed specificitysuch that it can bind to and cleave a variety of different nucleotidesequences, or the topoisomerase can be a site specific topoisomerase,which binds to and cleaves a specific nucleotide sequence. Thetopoisomerase also can be a type I topoisomerase, which cleaves onestrand of a ds nucleic acid molecule, or can be a type II topoisomerase,which cleaves both strands of a ds nucleic acid molecule. Where thetopoisomerase is a type II topoisomerase, cleavage is effected such thata linear ds nucleic acid molecule is produced, and istopoisomerase-charged at one or both ends. In certain such aspects, thestrand of the ds nucleic acid molecule that is complementary to thestrand containing the bound topoisomerase will form an overhangingsequence.

An advantage of performing a method of the invention is that theligation reaction performed by a topoisomerase occurs very quickly andover a wide range of temperatures. An additional advantage is thatrecombinant nucleic acid molecules generated according to the methods ofthe invention do not contain nicks at the sites where two nucleic acidmolecules are joined together. As such, the covalently linkedrecombinant nucleic acid molecules can be used directly in a subsequentprocedure, for example, as a substrate for an amplification reactionsuch as a polymerase chain reaction (PCR).

By way of example, a method of the invention can be performed bycontacting 1) a first nucleic acid molecule (which may be ss or ds)having a first end and a second end, wherein, at the first end or secondend or both, the first nucleic acid molecule has a topoisomeraserecognition site at or near the 3′ terminus; 2) at least a secondnucleic acid molecule (which may also be ss or ds) having a first endand a second end, wherein, at the first end or second end or both, theat least second double stranded nucleotide sequence has a topoisomeraserecognition site at or near the 3′ terminus; and 3) a site specifictopoisomerase, under conditions such that all components are in contactand the topoisomerase can effect its activity. The strand complementaryto that containing the topoisomerase recognition sequence may comprise a5′ hydroxyl group and, upon cleavage by the topoisomerase, may furthercomprise a 5′ overhanging sequence.

A method of the invention also can be performed by contacting 1) anucleic acid molecule (which may be ss or ds) having a first end and asecond end, wherein each of the first end and second end contains atopoisomerase recognition site at or near the 3′ terminus, and 2) a sitespecific topoisomerase, under conditions such that the components are incontact and the topoisomerase can effect its activity. For example, thetopoisomerase can be a type IB topoisomerase such as a Vacciniatopoisomerase or an S. cerevisiae topoisomerase. Such a method providesa means to prepare a covalently closed circular or supercoiled dsnucleic acid molecule.

A method of the invention also can be performed by contacting 1) a firstnucleic acid molecule (which may be ss or ds) having a first end and asecond end, wherein the first nucleic acid molecule has a topoisomeraserecognition site at or near the 5′ terminus of the first end or thesecond end or both; 2) at least a second nucleic acid molecule (whichmay also be ss or ds) having a first end and a second end, wherein theat least second double stranded nucleotide sequence has a topoisomeraserecognition site at or near the 5′ terminus of the first end or thesecond end or both; and 3) at least one site specific topoisomerase,under conditions such that all components are in contact and the atleast one topoisomerase can effect its activity. For example, thetopoisomerase can be a type IA topoisomerase such as an E. colitopoisomerase I or topoisomerase III, or eukaryotic topoisomerase III.Upon cleavage of a nucleic acid molecule, the topoisomerase preferablyis stably bound to the 5′ terminus. The 3′ terminus of the endcontaining the topoisomerase recognition site, or bound topoisomerase,can comprise a 3′ hydroxyl group, or can be modified to comprise a 3′hydroxyl group. Upon cleavage by the topoisomerase, the cleaved nucleicacid molecule may comprise a 3′ overhanging sequence.

The methods as exemplified herein can be performed using two or moresite specific topoisomerases, wherein the first, second or other nucleicacid substrates correspondingly have, at or near a 3′ terminus or 5′terminus of an end, a topoisomerase recognition site for one of the twoor more topoisomerases. The use of two or more topoisomerases, andcorresponding topoisomerase recognition sites, can facilitate thejoining of the nucleic acid molecules (which may be ss or ds) in apredetermined order, orientation, or combination thereof. Thus, it willbe recognized that, where a method of the invention is exemplified usinga topoisomerase, the method similarly can be performed using two or moretopoisomerases. In some cases, reference is made to the use of at leastone topoisomerase, although, unless indicated otherwise, the methods canbe performed using one, two, three or more topoisomerases, provided thesubstrate nucleic acid molecules contain the appropriate topoisomeraserecognition sites. Similar considerations are relevant totopoisomerase-charged nucleic acid substrates, in that thetopoisomerases can be the same or different.

In another embodiment, a method of the invention can be performed bycontacting 1) a first nucleic acid molecule (which may be ss or ds)having a first end and a second end, wherein the first nucleic acidmolecule has a topoisomerase recognition site at or near the 3′ terminusand a topoisomerase recognition site at or near the 5′ terminus of thefirst end or of the second end or of both ends; 2) at least a secondnucleic acid molecule (which may also be ss or ds) having a first endand a second end; and 3) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,etc.) site specific topoisomerases, under conditions such that allcomponents are in contact and each of the topoisomerases can effect itsactivity. Upon cleavage of the termini of the substrate first nucleicacid molecule by the topoisomerases, the 5′ terminus or the 3′ terminusof one or both ends can comprise an overhanging sequence, or can beblunt ended, or one end can contain an overhang and the second end canbe blunt ended. Where present, an overhanging sequence generally hassufficient complementarity to an overhanging sequence of the second (orother) nucleic acid molecule to allow for specific hybridization of thetwo molecules to each other.

Once nucleic acid molecules are joined by topoisomerase mediated joiningmethods of the invention, the resulting nucleic acid molecules may thenbe used in recombination reactions, such as those described elsewhereherein.

The number of different topoisomerases useful in such an embodiment willdepend, in part, on whether the first nucleic acid molecule containstopoisomerase recognition sites at only the first end or the second end,or contains topoisomerase recognition sites at both ends, and further,where the nucleic acid molecule contains topoisomerase recognition siteson both ends, whether at least the 3′ recognition sites or the 5′recognition sites are different. In addition, the method can beperformed such that one or more of the at least second nucleic acidmolecule also can contain a topoisomerase recognition site at or nearthe 3′ terminus and/or a topoisomerase recognition site at or near the5′ terminus of the first end or of the second end or of both ends,wherein the topoisomerase recognition sites at or near the 3′ terminusor the 5′ terminus or both of the other nucleic acid molecule can thesame as or different from the topoisomerase recognition sites in thefirst nucleic acid molecule. As such, the number of differenttopoisomerase further will depend on the number of different substratenucleic acid molecules being linked according to a method of theinvention.

An advantage of performing a method of the invention using a sitespecific topoisomerase is that the first nucleic acid molecule, thesecond nucleic acid molecule, and one or more additional nucleic acidmolecules (which may be ss or ds) can be covalently linked in apredetermined directional orientation. An additional advantage is that afunctional product can be selected in vitro by performing anamplification reaction using primers specific for the termini of thedesired covalently linked recombinant nucleic acid molecule. As such, acovalently linked recombinant nucleic acid molecule (which may be ss ords) generated according to a method of the invention can be useddirectly in further procedures, for example, for transfecting a cell, oras a template for performing amplification (e.g., PCR), a recombinationreaction (e.g., a recombination reaction such as those describedherein), an in vitro transcription reaction, or a coupledtranscription/translation reaction. Accordingly, the covalently linkedrecombinant nucleic acid molecule is useful, without furthermanipulation, for various purposes.

In an aspect of the invention, the first nucleic acid molecules, as wellas other nucleic acids used in methods of the invention, may be derivedfrom at least a first population of nucleic acid molecules, for example,from a cDNA library or a combinatorial library such as a combinatoriallibrary of synthetic oligonucleotides, and the second nucleic acidmolecules, as well as other nucleic acids used in methods of theinvention, may be derived from at least a second population of sourcenucleic acid molecules. According to such a method, linking of firstnucleic acid molecules with second nucleic acid molecules provides ameans to generate combinatorial populations of covalently linkedrecombinant nucleic acid molecules (which may be ss or ds). Inaccordance with such a method, one or more target nucleic acid moleculesalso can be linked with the recombinant nucleic acid molecules of thepopulation to produce additional populations. Such populations ofcombinatorial molecules can be further manipulated or analyzed, forexample, by protein expression and screening for fusion proteins havingdesirable characteristics.

In one embodiment, a method of the invention is performed such that thefirst nucleic acid molecule (which may be ss or ds), as well as othernucleic acids used in methods of the invention, comprises an openreading flame, for example, an isolated cDNA or coding sequence of agene, and a second nucleic acid molecule (which may be ss or ds)comprises a regulatory element such as a promoter, which can be operablycovalently linked to the 5′ end of the coding sequence such that thecoding sequence can be transcribed therefrom. A second nucleic acidmolecule, as well as other nucleic acids used in methods of theinvention, also can comprise two or more regulatory elements, forexample, a promoter (e.g., a GAL4 promoter), an operator (e.g., a tetoperator, a galactose operon operator, a lac operon operator, and thelike), an internal ribosome entry site and an ATG initiator methioninecodon, in operative linkage with each other, which can be operablycovalently linked to the 5′ end of a first nucleic acid moleculecomprising a coding sequence according to a method of the invention.Such a method can further include contacting a third nucleic acidmolecule (which may be ss or ds) comprising, for example, apolyadenylation signal, which can be operably covalently linked to the3′ end of the coding sequence. Such a method can be useful forgenerating an expressible nucleic acid molecule, which can betranscribed, translated, or both as a functional unit. In addition, oralternatively, a nucleic acid molecule encoding a detectable marker, forexample, an epitope tag, can be operably linked to a first or second (orother) nucleic acid molecule(s) according to a method of the invention.The generation of a recombinant nucleic acid molecule (which may be ssor ds) having a desired directional orientation of the nucleotidesequences in such a construct may be facilitated, for example, byincluding complementary 5′ overhanging sequences at the termini of thenucleic acid molecules to be covalently linked together by thetopoisomerase.

In another embodiment, a method of the invention is performed such thatat least the first nucleic acid molecule or the at least second nucleicacid molecule, as well as other nucleic acids used in methods of theinvention, is one of a plurality of nucleotide sequences, for example, acDNA library, a combinatorial library of nucleotide sequences, or avariegated population of nucleotide sequences. In another embodiment, amethod of the invention includes further contacting a generatedcovalently linked ds recombinant nucleic acid molecule (e.g., arecombinant nucleic acid molecule which is covalently linked in one orboth strands) with a PCR primer pair, and amplifying all or a portion ofthe covalently linked recombinant nucleic acid molecule. In addition togenerating a large amount of product, the amplification reaction can beselective for constructs comprising a desired covalently linked dsrecombinant nucleic acid molecule, particularly where the nucleic acidmolecules to be covalently linked comprise complementary overhangingsequences. As such, a method of the invention provides an in vitroselection means that is suitable for high throughput analysis.

A method of the invention is also exemplified by contacting 1) a firstnucleic acid molecule (which may be ss or ds) having a first end and asecond end, wherein, at the first end or second end or both, the firstnucleic acid molecule has a topoisomerase covalently bound to the 3′terminus (“topoisomerase-charged”); and 2) at least a secondtopoisomerase-charged nucleic acid molecule (which may be ss or ds).Preferably, the topoisomerase-charged nucleic acid molecules contain a5′ hydroxyl group at the ends containing the bound topoisomerase,although 5′ hydroxy groups also can be generated using a phosphatase.The methods of the invention can be performed using only a first nucleicacid molecule and a second nucleic acid molecule, or can include athird, fourth or more nucleic acid molecules (which may be ss or ds) asdesired, wherein each nucleotide sequence is as defined. A first orsecond (or other) nucleic acid molecule independently can have atopoisomerase covalently bound to a 3′ terminus of one end or at bothends of the nucleotide sequence, and, unless indicated otherwise, thefirst and second (or other) nucleic acid molecules can be the same orcan be different. In certain such aspects, at least one of the nucleicacid molecules used in the methods described herein will comprise atleast one recombination site. Further, nucleic acid molecules generatedby methods described above may be used in recombination reactions, suchas those described elsewhere herein.

Methods of the invention are further exemplified by contacting 1) afirst nucleic acid molecule (which may be ss or ds) having a first endand a second end, wherein, at the first end or second end or both, thefirst nucleic acid molecule has a topoisomerase covalently bound to a 5′terminus (i.e., a topoisomerase-charged 5′ terminus); and 2) at least asecond topoisomerase-charged nucleic acid molecule (which may be ss ords) comprising at least one topoisomerase-charged 5′ terminus. Thetopoisomerase-charged nucleic acid molecules can contain a 3′ hydroxylgroup at the ends containing the bound topoisomerase, or a 3′ hydroxylgroup can be generated using a phosphatase. As disclosed herein, such amethod can be performed using only a first nucleic acid molecule and asecond nucleic acid molecule, or can include a third, fourth or morenucleic acid molecules (which may be ss or ds) as desired, wherein eachnucleotide sequence is as defined, including comprising at least onetopoisomerase-charged 5′ terminus. A first or second (or other) nucleicacid molecule independently can have a topoisomerase covalently bound toa 5′ terminus of one end or at both ends of the nucleic acid molecule,and, unless indicated otherwise, the first and second (or other) nucleicacid molecules can be the same or can be different. In certain suchaspects, at least one of the nucleic acid molecules used in the methodsdescribed herein will comprise at least one recombination site. Further,nucleic acid molecules generated by methods described above andelsewhere herein may also be used in recombination reactions, such asthose described elsewhere herein.

A method of the invention is additionally exemplified by contacting 1) afirst nucleic acid molecule having a first end and a second end,wherein, at the first end or second end or both, the first nucleic acidmolecule has a first topoisomerase covalently bound to the 5′ terminusand a second topoisomerase covalently bound to the 3′ terminus of thefirst end or the second end or both (i.e., one or both ends contain atopoisomerase charged 5′ terminus and a topoisomerase-charged 3′terminus); and 2) at least a second nucleic acid molecule, which,preferably, has or can be made to have hydroxyl groups at the 5′terminus and 3′ terminus of an end to be covalently linked to an end ofthe first nucleic acid molecule containing the topoisomerases. Themethod also can be performed wherein either the 5′ terminus or 3′terminus of the end containing a topoisomerase-charged 3′ terminus ortopoisomerase-charged 5′ terminus, respectively, contains atopoisomerase recognition site, wherein the method further includescontacting the components with a topoisomerase that can effect itsactivity with respect to the topoisomerase recognition site. In certainsuch aspects, at least one of the nucleic acid molecules used in themethods described herein will comprise at least one recombination site.Further, nucleic acid molecules generated by methods described above andelsewhere herein may also be used in recombination reactions, such asthose described elsewhere herein.

Such a method of the invention can be performed using only a firstnucleic acid molecule and a second nucleic acid molecule, or can includea third, fourth or more nucleic acid molecule as desired, wherein thenucleic acid molecules are as defined for the first nucleic acidmolecule, the second nucleic acid molecule, or a combination thereof. Afirst or second (or other) nucleic acid molecule independently can, butneed not, have one or more topoisomerases covalently bound to a 5′terminus, 3′ terminus, or both 5′ and 3′ termini of the second end(i.e., the undefined end). Further, one or more of these nucleic acidmolecules may additionally comprise one or more recombination sites.Unless indicated otherwise, the first and second (or other) nucleic acidmolecules can be the same or can be different.

The present invention further relates to a method of generating acovalently linked ds recombinant nucleic acid molecule by 1) amplifyinga portion of a first nucleic acid molecule using a PCR primer pair,wherein at least one primer of the primer pair encodes a complement of atopoisomerase recognition site, and, optionally, of one or morerecombination sites, thereby producing an amplified first nucleic acidmolecule having a first end and a second end, wherein the first end orsecond end or both has a topoisomerase recognition site at or near the3′ terminus; and 2) contacting a) the amplified first nucleic acidmolecule; b) at least a second nucleic acid molecule having a first endand a second end, wherein the first end or second end or both has atopoisomerase recognition-site, or cleavage product thereof, at or nearthe 3′ terminus and has, or can be made to have, a hydroxyl group at the5′ terminus of the same end; and c) a site specific topoisomerase, underconditions such that the topoisomerase can cleave the end of theamplified first nucleic acid molecule having a topoisomerase recognitionsite and the end (or ends) of the at least second nucleic acid moleculehaving a topoisomerase recognition site, and can effect its ligatingactivity. The PCR primer that encodes a complement of topoisomeraserecognition site can have a hydroxyl group at its 5′ terminus, or theamplified first nucleic acid molecule generated using the primer can becontacted with a phosphatase to generate a hydroxyl group at its 5′terminus. The PCR primer encoding the complement of a topoisomeraserecognition site also can comprise a nucleotide sequence at its 5′terminus such that, upon cleavage by a site specific topoisomerase of afirst nucleic acid molecule amplified using the primer, the nucleic acidmolecule contains a 5′ overhanging sequence, which is complementary to a5′ overhanging sequence of a second (or other) nucleic acid molecule towhich the first nucleic acid molecule is to be covalently linkedaccording to a method of the invention. In certain such aspects, atleast one of the nucleic acid molecules used in the methods describedherein will comprise at least one recombination site. Further, nucleicacid molecules generated by methods described above and elsewhere hereinmay also be used in recombination reactions, such as those describedelsewhere herein.

The present invention also relates to a method of generating acovalently linked ds recombinant nucleic acid molecule by 1) amplifyinga portion of a first nucleic acid molecule using a PCR primer pair,wherein at least one primer of the primer pair encodes a topoisomeraserecognition site, and, optionally, one or more recombination sites,thereby producing an amplified first nucleic acid molecule having afirst end and a second end, wherein the first end or second end or bothhas a topoisomerase recognition site at or near the 5′ terminus; and 2)contacting a) the amplified first nucleic acid molecule; b) at least asecond nucleic acid molecule having a first end and a second end,wherein the first end or second end or both has a topoisomeraserecognition site at or near the 5′ terminus and has, or can be made tohave, a hydroxyl group at the 3′ terminus of the same end; and c) atleast one site specific topoisomerase, under conditions such that the atleast one topoisomerase can cleave the end of the amplified firstnucleic acid molecule having a topoisomerase recognition site and theend (or ends) of the at least second nucleic acid molecule having atopoisomerase recognition site, and can effect its ligating activity.The amplified first nucleic acid molecule generally has a hydroxyl groupat the 3′ terminus of the end containing the topoisomerase recognitionsite, or can be modified to contain such a 3′ hydroxyl group. The PCRprimer encoding the topoisomerase recognition site can further comprisea nucleotide sequence at its 5′ terminus, i.e., 5′ to the topoisomeraserecognition site, such that, upon cleavage of the amplified firstnucleic acid molecule by a site specific topoisomerase, the nucleic acidmolecule contains a 3′ overhanging sequence, which is complementary to a3′ overhanging sequence of a second (or other) nucleic acid molecule towhich the first nucleic acid molecule is to be covalently linkedaccording to a method of the invention. In certain such aspects, atleast one of the nucleic acid molecules used in the methods describedherein will comprise at least one recombination site. Further, nucleicacid molecules generated by methods described above and elsewhere hereinmay also be used in recombination reactions, such as those describedelsewhere herein.

The present invention further relates to a method of generating acovalently linked ds recombinant nucleic acid molecule by 1) amplifyinga portion of a first nucleic acid molecule using a PCR primer pair,wherein at least one primer of the primer pair includes a topoisomeraserecognition site, a nucleotide sequence complementary to a topoisomeraserecognition site, such that PCR introduces a functional recognition sitein the opposite strand (see primer sequences in FIG. 9D), and,optionally, a recombination site, thereby producing an amplified firstnucleic acid molecule having a first end and a second end, wherein theamplified first nucleic acid molecule has a topoisomerase recognitionsite at or near the 5′ terminus and a topoisomerase recognition site ator near the 3′ terminus of the first end or of the second end or of bothends; and 2) contacting a) the amplified first nucleic acid molecule; b)at least a second nucleic acid molecule having a first end and a secondend, wherein the second nucleic acid molecule has, or can be made tohave, a 5′ hydroxyl group and a 3′ hydroxyl group at the first end or atsecond end or at both ends; and c) at least two site specifictopoisomerases, under conditions such that i) at least one topoisomerasecan cleave the topoisomerase recognition site at or near the 5′ terminusof the end of the amplified first nucleic acid molecule, and can effectits ligating activity, and ii) at least one topoisomerase can cleave thetopoisomerase recognition site at or near the 3′ terminus of the end ofthe amplified first nucleic acid molecule, and can effect its ligatingactivity. Accordingly, the present invention provides a nucleic acidmolecule containing, at one or both ends, a topoisomerase recognitionsite at or near the 5′ terminus and a topoisomerase recognition site ator near the 3′ terminus. In addition, the invention provides such anucleic acid molecule, which is topoisomerase charged at the 5′ terminusor the 3′ terminus or both. In certain such aspects, at least one of thenucleic acid molecules used in the methods described herein willcomprise at least one recombination site. Further, nucleic acidmolecules generated by methods described above and elsewhere herein mayalso be used in recombination reactions, such as those describedelsewhere herein.

The present invention further relates to an oligonucleotide containingat least one recognition site of one or more type IA site specifictopoisomerases, at least one nucleotide sequence complementary to arecognition site of one or more type IB site specific topoisomerasesand, optionally, at least one recombination site. Such anoligonucleotide is useful, for example, as a primer for a primerextension reaction or as one of a primer pair for performing anamplification reaction such as PCR. Such an oligonucleotide, referred toherein as an oligonucleotide primer, can be one of a primer pair, whichcan be useful for generating a ds nucleic acid amplification productthat contains, at one end, a type IA topoisomerase recognition site ator near the 5′ terminus and, at the same end, a type IB topoisomeraserecognition site at or near the 3′ terminus. The oligonucleotide primercan further contain a nucleotide sequence encoding (or complementary to)any other nucleotide sequence or peptide of interest, for example, arestriction endonuclease recognition site, a peptide tag, and, ifdesired, one or more additional type IA or type IB topoisomeraserecognition sites, thereby allowing selection of one or more convenientor readily available topoisomerases for practicing a method of theinvention. The oligonucleotide primer can further comprise a nucleotidesequence at its 5′ terminus, i.e., 5′ to the type IA topoisomeraserecognition site or to the nucleotide sequence complementary to the typeIB topoisomerase recognition site, such that, upon cleavage of theamplified first nucleic acid molecule by a site specific topoisomerase,the nucleic acid molecule contains a 3′ or 5′ overhanging sequence,respectively, which is complementary to a 3′ or 5′ overhanging sequence,respectively, of a second (or other) nucleic acid molecule to which thefirst nucleic acid molecule is to be covalently linked according to amethod of the invention, or the oligonucleotide primer can be designedsuch that, upon cleavage of an amplified nucleic acid molecule generatedtherefrom, a blunt end topoisomerase charged nucleic acid molecule isgenerated.

The invention further relates to an oligonucleotide which contains atleast one topoisomerase recognition site, or a nucleotide sequencecomplementary thereto, and at least one recombination site. Such anoligonucleotide may be used as described above, for example as onemember of a primer pair.

Oligonucleotides of the invention will often be between 15-20, 15-30,15-50, 20-30, 20-50, 30-40, 30-50, 30-80, 30-100, 40-50, 40-70, 40-80,40-100, 50-60, 50-80, 50-100, 15-80, 15-100, or 20-100 (or the like)nucleotides in length.

The present invention also provides a primer pair, which includes atleast-one oligonucleotide primer as defined above, wherein one of theprimers is useful as a forward primer and the primer is useful as areverse primer in an amplification reaction. The second primer in such aprimer pair can, but need not, include a type IA topoisomeraserecognition site, a nucleotide sequence complementary to a type IBtopoisomerase recognition site, or both, and can include any othernucleotide sequence of interest and/or at least one recombination site.In one embodiment, the primer pair includes two oligonucleotide primersof the invention, wherein one oligonucleotide primer is useful as aforward primer and the second oligonucleotide primer is useful as areverse primer, such a primer pair being useful, for example, forgenerating a nucleic acid molecule amplification product havingtopoisomerase recognition sites at both termini of both ends and/or oneor more recombination sites, wherein the type IA or type IB or bothtopoisomerase recognition sites at the termini are the same ordifferent.

Accordingly, the present invention further relates to a nucleic acidmolecule, which has a first end and a second end, and which contains atype IA topoisomerase recognition site at or near the 5′ terminus and atype IB topoisomerase recognition site at or near the 3′ terminus of thefirst end or of the second end or of both ends. In addition, the presentinvention provides a nucleic acid molecule as defined above, exceptwherein the nucleic acid molecule is a topoisomerase charged molecule,comprising a stably bound type IA topoisomerase or a type IBtopoisomerase or both, at one or both ends, as desired. These nucleicacid molecules may further comprise one or more recombination sites.

In one embodiment, the first nucleic acid molecule, as well as othernucleic acids used in methods of the invention, comprises an expressiblenucleotide sequence which encodes molecules such as a polypeptide (whichmay be, e.g., a polypeptide with an intein), an antisense nucleotidesequence, interference RNA (i.e., “RNAi”) molecule(s), a ribozyme, atransfer RNA (i.e., a tRNA, including but not limited to a supressortRNA), a triplexing nucleotide sequence, and the like, and the second(or other) nucleic acid molecule comprises a transcription regulatoryelement such as a promoter (e.g., a GAL4 operator), an operator (e.g., atet operator, a galactose operon operator, a lac operon operator, andthe like), an enhancer, a silencer, a translation start site, or apolyadenylation signal, or encodes a translation regulatory element suchas an initiator methionine, a STOP codon, a cell compartmentalizationdomain, a homology domain, or the like, or a combination thereof inoperative linkage. A second (or other) nucleic acid molecule, as well asother nucleic acids used in methods of the invention, which can be anamplified second (or other) nucleic acid molecule prepared as for theamplified first nucleic acid molecule, also can comprise one or moremultiple cloning sites (“MCS”), a detectable label, for example, anenzyme, a substrate for an enzyme, a fluorescent compound, a luminescentcompound, a chemiluminescent compound, a radionuclide, a paramagneticcompound, and biotin; or can include a tag, which can be anoligonucleotide tag or can be a peptide tag, for example, apolyhistidine tag, a V5 epitope, or a myc epitope.

In another embodiment, a method of the invention is performed using afirst nucleic acid molecule that encodes a polypeptide (e.g., apolypeptide which contains an intein), or a domain thereof, and a second(or other) nucleic acid molecule that encodes a transcription activationdomain or a DNA binding domain. Such a method can be used to generatecovalently linked ds recombinant nucleic acid molecules that encodechimeric polypeptides useful for performing a two hybrid assay system,particularly a high throughput two hybrid assay. In still anotherembodiment, the first nucleic acid molecules comprises a plurality ofnucleotide sequences, which can be a cDNA library, a combinatoriallibrary of nucleotide sequences, a variegated population of nucleotidesequences, or the like.

A method of the invention provides a means to generate a covalentlylinked ds recombinant nucleic acid molecule useful for site specificinsertion into a target genomic DNA sequence. The target genomic DNAsequence can be any genomic sequence, particularly a gene, andpreferably a gene for which some or all of the nucleotide sequence isknown. The method can be performed utilizing two sets of PCR primerpairs and a nucleic acid molecule. The nucleic acid molecule has a firstend and a second end and encodes a polypeptide, for example, aselectable marker, wherein the nucleic acid molecule comprises atopoisomerase recognition site or cleavage product thereof at the 3′terminus of each end and, optionally, a hydroxyl group at the 5′terminus of each end, and wherein, preferably, the 5′ termini compriseoverhanging sequences, which are different from each other. Similarly,the nucleic acid molecule can comprise a topoisomerase recognition siteor cleavage product thereof at or near the 5′ terminus of one or bothends and, optionally, a hydroxyl group at the 3′ terminus of one or bothend, and wherein one or both the 3′ termini can comprise overhangingsequences, which can be the same as or, preferably, different from eachother; or the 5′ terminus and 3′ terminus of one or both ends of thenucleic acid molecule each can comprise a topoisomerase recognition siteor cleavage product thereof (see FIG. 11). In certain such aspects, atleast one of the nucleic acid molecules used in the methods describedherein will comprise at least one recombination site. Further, nucleicacid molecules generated by methods described above and elsewhere hereinmay also be used in recombination reactions, such as those describedelsewhere herein.

The two sets of PCR primer pairs will generally be selected such that,in the presence of an appropriate DNA polymerase such as Taq polymeraseand a template comprising the sequences to be amplified, the primersamplify portions of a genomic DNA sequence that are upstream (andadjacent to) and downstream (and adjacent to) of the target site forinsertion of the polypeptide (e.g., selectable marker). The sets of PCRprimer pairs also are designed such that the amplification productscontain a topoisomerase recognition site at least at the end to becovalently linked to the selectable marker, including at or near the 5′terminus, or the 3′ terminus, or both, as appropriate for the particularmethod of the invention being practiced. As such, the first PCR primerpair can include, for example, 1) a first primer, which comprises, in anorientation from 5′ to 3′, a nucleotide sequence complementary to a 5′overhanging sequence of the end of the selectable marker to which theamplification product is to be covalently linked, a nucleotide sequencecomplementary to a topoisomerase recognition site, such that PCRintroduces a functional recognition site in the opposite strand (seeprimer sequences in FIG. 9D), and a nucleotide sequence complementary toa 3′ sequence of a target genomic DNA sequence; and 2) a second primer,which comprises a nucleotide sequence of the target genomic DNA upstreamof the 3′ sequence to which the first primer is complementary. Thesecond PCR primer pair includes 1) a first primer, which comprises, from5′ to 3′, a nucleotide sequence complementary to the 5′ overhangingsequence of the end of the selectable marker to which it is to becovalently linked, a nucleotide sequence complementary to atopoisomerase recognition site, such that PCR introduces a functionalrecognition site in the opposite strand (see primer sequences in FIG.9D), and a nucleotide sequence of a 5′ sequence of a target genomic DNAsequence, wherein the 5′ sequence of the target genomic DNA isdownstream of the 3′ sequence of the target genomic DNA to which thefirst primer of the first PCR primer pair is complementary; and 2) asecond primer, which comprises a nucleotide sequence complementary to a3′ sequence of the target genomic DNA that is downstream of the 5′sequence of the target genomic DNA contained in the first primer.

Upon contact of the nucleic acid molecule comprising the selectablemarker, the PCR amplification products, and at least one topoisomerase,a covalently linked ds recombinant nucleic acid molecule is generatedaccording to a method of the invention. The generated ds recombinantnucleic acid molecule is useful for performing homologous recombinationin a genome, for example, to knock-out the function of a gene in a cell,or to confer a novel phenotype on the cell containing the generated dsrecombinant nucleic acid molecule. The method can further be used toproduce a transgenic non-human organism having the generated recombinantnucleic acid molecule stably maintained in its genome.

The present invention also relates to compositions prepared according tothe methods of the invention, and to compositions useful for practicingthe methods. Such compositions can include one or more reactants used inthe methods of the invention and/or one or more ds recombinant nucleicacid molecules produced according to a method of the invention. Suchcompositions can include, for example, one or more nucleic acidmolecules with one or more topoisomerase recognition sites; one or moretopoisomerase-charge nucleic acid molecules; one or more nucleic acidmolecules comprising one or more recombination sites; one or moreprimers useful for preparing a nucleic acid molecule containing atopoisomerase recognition site at one or both termini of one or bothends of an amplification product prepared using the primer; one or moretopoisomerases; one or more substrate nucleic acid molecules, including,for example, nucleotide sequences encoding tags, markers, regulatoryelements, or the like; one or more covalently linked ds recombinantnucleic acid molecules produced according to a method of the invention;one or more cells containing or useful for containing a nucleic acidmolecule, primer, or recombinant nucleic acid molecule as disclosedherein; one or more polymerases for performing a primer extension oramplification reaction; one or more reaction buffers; and the like. Inone embodiment, a composition of the invention comprises two or moredifferent topoisomerase-charged nucleic acid molecules and/or two ormore different recombination sites. The composition can further compriseat least one topoisomerase. A composition of the invention also cancomprise a site specific topoisomerase and a covalently linked dsrecombinant nucleic acid molecule, wherein the recombinant nucleic acidmolecule contains at least one topoisomerase recognition site for thesite specific topoisomerase in each strand, and wherein a topoisomeraserecognition site in one strand is within about 100 nucleotides of atopoisomerase recognition site in the complementary strand, generallywithin about five, ten, twenty or thirty nucleotides.

Product molecules produced by methods of the invention may comprise anycombination of starting molecules (or portions thereof) and can be anysize and be in any form (e.g., circular, linear, supercoiled, etc.),depending on the starting nucleic acid molecule or segment, the locationof the recombination sites on the molecule, and the order ofrecombination of the sites.

Any of the product molecules of the invention may be furthermanipulated, analyzed or used in any number of standard molecularbiology techniques or combinations of such techniques (in vitro or invivo). These techniques include sequencing, amplification, nucleic acidsynthesis, protein or peptide expression (for example, fusion proteinexpression, antibody expression, hormone expression etc.),protein-protein interactions (2-hybrid or reverse 2-hybrid analysis),homologous recombination or gene targeting, and combinatorial libraryanalysis and manipulation. The invention also relates to cloning thenucleic acid molecules of the invention (preferably by recombination)into one or more vectors or converting the nucleic acid molecules of theinvention into a vector by the addition of certain functional vectorsequences (e.g., origins of replication). In one aspect, recombinationand/or topoisomerase-mediated joining is accomplished in vitro andfurther manipulation or analysis is performed directly in vitro. Thus,further analysis and manipulation will not be constrained by the abilityto introduce the molecules of the invention into a host cell and/ormaintained in a host cell. Thus, less time and higher throughput may beaccomplished by further manipulating or analyzing the molecules of theinvention directly in in vitro, although in vitro analysis ormanipulation can be done after passage through host cells or can be donedirectly in vivo (while in the host cells).

Nucleic acid synthesis steps, according to the invention, may comprise:

(a) mixing a nucleic acid molecule of interest or template with one ormore primers and one or more nucleotides to form a mixture; and

(b) incubating said mixture under conditions sufficient to synthesize anucleic acid molecule complementary to all or a portion of said moleculeor template.

The synthesized molecule may then be used as a template for furthersynthesis of a nucleic acid molecule complementary to all or a portionof the first synthesized molecule. Accordingly, a double strandednucleic acid molecule (e.g., DNA) may be prepared. Preferably, suchsecond synthesis step is preformed in the presence of one or moreprimers and one or more nucleotides under conditions sufficient tosynthesize the second nucleic acid molecule complementary to all or aportion of the first nucleic acid molecule. Typically, synthesis of oneor more nucleic acid molecules is performed in the presence of one ormore polymerases (preferably DNA polymerases which may be thermostableor mesophilic), although reverse transcriptases may also be used in suchsynthesis reactions. Accordingly, the nucleic acid molecules used astemplates for the synthesis of additional nucleic acid molecules may beRNA, mRNA, DNA or non-natural or derivative nucleic acid molecules.Nucleic acid synthesis, according to the invention, may be facilitatedby incorporating one or more primer sites into the product moleculesthrough the use of starting nucleic acid molecules containing suchprimer sites. Thus, by the methods of the invention, primer sites may beadded at one or a number of desired locations in the product molecules,depending on the location of the primer site within the startingmolecule and the order of addition of the starting molecule in theproduct molecule.

Sequencing steps, according to the invention, may comprise:

(a) mixing a nucleic acid molecule to be sequenced with one or moreprimers, one or more nucleotides and one or more termination agents toform a mixture;

(b) incubating said mixture under conditions sufficient to synthesize apopulation of molecules complementary to all or a portion of saidmolecules to be sequenced; and

(c) separating said population to determine the nucleotide sequence ofall or a portion of said molecule to be sequenced.

Such sequencing steps are preferably performed in the presence of one ormore polymerases (e.g., DNA polymerases and/or reverse transcriptases)and one or more primers. Preferred terminating agents for sequencinginclude derivative nucleotides such as dideoxynucleotides (ddATP, ddTTP,ddGTP, ddCTP and derivatives thereof). Nucleic acid sequencing,according to the invention, may be facilitated by incorporating one ormore sequencing primer sites into the product molecules through the useof starting nucleic acid molecules containing such primer sites. Thus,by the methods of the invention, sequencing primer sites may be added atone or a number of desired locations in the product molecules, dependingon the location of the primer site within the starting molecule and theorder of addition of the starting molecule in the product molecule.

Protein expression steps, according to the invention, may comprise:

(a) obtaining a nucleic acid molecule to be expressed which comprisesone or more expression signals; and

(b) expressing all or a portion of the nucleic acid molecule undercontrol of said expression signal thereby producing a peptide or proteinencoded by said molecule or portion thereof

In this context, the expression signal may be said to be operably linkedto the sequence to be expressed. The protein or peptide expressed ispreferably expressed in a host cell (in vivo), although expression maybe conducted in vitro using techniques well known in the art. Uponexpression of the protein or peptide, the protein or peptide product mayoptionally be isolated or purified. Moreover, the expressed protein orpeptide may be used in various protein analysis techniques including2-hybrid interaction, protein functional analysis andagonist/antagonist-protein interactions (e.g., stimulation or inhibitionof protein function through drugs, compounds or other peptides). Thenovel and unique hybrid proteins or peptides (e.g., fusion proteins)produced by the invention and particularly from expression of thecombinatorial molecules of the invention may generally be useful fortherapeutics. Protein expression, according to the invention, may befacilitated by incorporating one or more transcription or translationsignals or regulatory sequences, start codons, termination signals,splice donor/acceptor sequences (e.g., intronic sequences) and the likeinto the product molecules through the use of starting nucleic acidmolecules containing such sequences. Thus, by the methods of theinvention, expression sequences may be added at one or a number ofdesired locations in the product molecules, depending on the location ofsuch sequences within the starting molecule and the order of addition ofthe starting molecule in the product molecule.

Homologous recombination, according to the invention, may comprise:

(a) mixing at least a first nucleic acid molecule of the invention(which is preferably a product molecule) comprising one or morerecombination sites and/or one or more toposiomerase recognition siteswith at least one target nucleic molecule, wherein said first and targetmolecules have one or more homologous sequences; and

(b) causing said first and target nucleic acid molecules to recombine byhomologous recombination. One example of a nucleic acid construct thatcan be used for homologous recombination is depicted in FIG. 37. Theinvention further includes methods for preparing nucleic acid moleculeswhich can be used for homologous recombination, and nucleic acidmolecules prepared by such methods, as well as cells which haveundergone homologous recombination according to methods of theinvention.

Such homologous recombination may occur in vitro, but preferably isaccomplished in vivo (e.g., in a host cell). Preferably, homologousrecombination causes transfer of all or a portion of a nucleic acidmolecule of the invention containing recombination sites (the firstnucleic acid molecule) into one or more positions of the target nucleicacid molecule containing homologous sequences. Selection of suchhomologous recombination may be facilitated by positive or negativeselection (e.g., using selectable markers) to select for a desiredproduct and/or against an undesired product. In a preferred aspect, thenucleic acid molecule of the invention comprises at least one selectablemarker and at least two sequences which are homologous to the targetmolecule. Preferably, the first molecule comprises at least twohomologous sequences flanking at least one selectable marker.

The present invention thus facilitates construction of gene targetingnucleic acid molecules or vectors which may be used to knock-out ormutate a sequence or gene of interest (or alter existing sequences, forexample to convert a mutant sequence to a wild type sequence),particularly genes or sequences within a host or host cells such asanimal, plant, human, insect, bacteria, and the like or sequences ofadventitious agents such as viruses within such host or host cells. Suchgene targeting may preferably comprise targeting a sequence on thegenome of such host cells. Such gene targeting may be conducted in vitroor in vivo. Thus, in a preferred aspect, the invention relates to amethod of targeting or mutating a sequence or a gene comprising:

(a) obtaining at least one nucleic acid molecule of the inventioncomprising one or more recombination sites and/or one or moretopoisomerase recognition sites (and preferably one or more selectablemarkers), wherein said molecule comprises one or more sequenceshomologous to the target gene or sequence of interest (said one or morehomologous sequences preferably flank one or more selectable markers onthe molecule of the invention); and

(b) contacting said molecule with one or more target genes or sequencesof interest under conditions sufficient to cause homologousrecombination at one or more sites between said target sequence or geneof interest and said molecule of the invention, thereby causinginsertion of all or a portion of the molecule of the invention withinthe target sequence or gene.

Such targeting method may cause deletion, inactivation or partialinactivation of the sequence or target gene such that an expressionproduct (typically a protein or peptide) normally expressed by suchsequence is not produced or produced at a higher or lower level or tothe extent produced is has an altered protein sequence which may resultin more or less activity or in an inactive or partially inactiveexpression product. The selectable marker preferably present on themolecule of the invention facilitates selection of candidates (forexample host cells) in which the homologous recombination event wassuccessful. Thus, the present invention provides a method to producehost cells, tissues, organs, and animals (e.g., transgenic animals)containing the modified gene or sequence produced by the targetingmethods of the invention. The modified sequence or gene preferablycomprises at least one recombination site and/or at least one selectablemarker provided by the molecule of the invention.

Thus, the present invention more specifically relates to a method oftargeting or mutating a sequence or a gene comprising:

(a) obtaining at least one nucleic acid molecule of the inventioncomprising one or more recombination sites, at least one selectablemarker flanked by one or more sequences homologous to the target gene orsequence of interest and, optionally, one or more topoisomeraserecognition sites;

(b) contacting said molecule with one or more target genes or sequencesof interest under conditions sufficient to cause homologousrecombination at one or more sites between said target sequence or geneof interest and said molecule, thereby causing insertion of all or aportion of the molecule of the invention (and preferably causinginsertion of at least one selectable marker and/or at least onerecombination site) within the target sequence or gene; and

(c) optionally selecting for said sequence or gene comprising all or aportion of the molecule of the invention or for a host cell containingsaid gene or sequence containing all or a portion of said molecule ofthe invention.

In another aspect of the invention, recombination sites introduced intotargeted sequences according to the invention may be used to excise orremove all or a portion of the molecule inserted into the targetsequence. Thus, the invention allows for in vitro or in vivo removal ofsuch sequences and thus may allow for reactivation of the target gene orsequence. In some embodiments, after identification and isolation of asequence containing the alterations introduced as above, a selectablemarker present on the molecule of the present invention may be removed.

The present invention also provides methods for cloning the starting orproduct nucleic acid molecules of the invention into one or more vectorsor converting the product molecules of the invention into one or morevectors. In one aspect, the starting molecules are recombined to makeone or more product molecules and such product molecules are cloned,(preferably by recombination) into one or more vectors. In anotheraspect, the starting molecules are cloned directly into one or morevectors such that a number of starting molecules are joined within thevector, thus creating a vector containing the product molecules of theinvention. In another aspect, the starting molecules are cloned directlyinto one or more vectors such that the starting molecules are not joinedwithin the vector (i.e., the starting molecules are separated by vectorsequences). In yet another aspect, a combination of product moleculesand starting molecules may be cloned in any order into one or morevectors, thus creating a vector comprising a new product moleculeresulting from a combination of the original starting and productmolecules.

Thus, the invention relates to a method of cloning comprising:

(a) obtaining at least one nucleic acid molecule of the inventioncomprising one or more recombination sites and/or one or moretopoisomerase recognition sites; and

(b) transferring all or a portion of said molecule into one or morevectors. The invention further includes vectors prepared by suchmethods, compositions comprising these vectors, and methods using thesevectors.

Such vectors will often comprise one or more recombination sites and/orone or more topoisomerase recognition sites, and the transfer of themolecules into such vectors is preferably accomplished by recombinationbetween one or more sites on the vectors and one or more sites on themolecules of the invention. In another aspect, the product molecules ofthe invention may be converted to molecules which function as vectors byincluding the necessary vector sequences (e.g., origins of replication).Thus, according to the invention, such vectors sequences may beincorporated into the product molecules through the use of startingmolecules containing such sequences. Such vector sequences may be addedat one or a number of desired locations in the product molecules,depending on the location of the sequence within the starting moleculeand the order of addition of the starting molecules in the productmolecule. The product molecule containing the vector sequences may be inlinear form or may be converted to a circular or supercoiled form bycausing recombination of recombination sites within the product moleculeor by a topoisomerase-mediated joining reaction. Often, circularizationof such product molecule is accomplished by recombining recombinationsites at or near both termini of the product molecule.

The vector sequences used in the invention may comprise one or a numberof elements and/or functional sequences and/or sites (or combinationsthereof) including one or more sequencing or amplification primer sites,one or more multiple cloning sites, one or more selectable markers(e.g., toxic genes, antibiotic resistance genes, selectable markersetc.), one or more transcription or translation sites or signals, one ormore transcription or translation termination sites, one or moretopoisomerase recognition sites, one or more topoisomerases, one or moreorigins of replication, one or more recombination sites (or portionsthereof), etc. The vector sequences used in the invention may alsocomprise stop codons which may be suppressed to allow expression ofdesired fusion proteins as described herein. Thus, according to theinvention, vector sequences may be used to introduce one or more of suchelements, functional sequences and/or sites into any of the nucleic acidmolecule of the invention, and such sequences may be used to furthermanipulate or analyze any such nucleic acid molecule cloned into suchvectors. For example, primer sites provided by a vector (preferablylocated on both sides of the insert cloned in such vector) allowsequencing or amplification of all or a portion of a product moleculecloned into the vector. Additionally, transcriptional or regulatorysequences contained by the vector allows expression of peptides,polypeptides or proteins encoded by all or a portion of the productmolecules cloned to the vector. Likewise, genes, portion of genes orsequence tags (such as GUS, GST, GFP, His tags, epitope tags and thelike) provided by the vectors allow creation of populations of genefusions with the product molecules cloned in the vector or allowsproduction of a number of peptide, polypeptide or protein fusionsencoded by the sequence tags provided by the vector in combination withthe product sequences cloned in such vector. Such genes, portions ofgenes or sequence tags may be used in combination with optionallysuppressed stop codons to allow controlled expression of fusion proteinsencoded by the sequence of interest being cloned into the vector and thevector supplied gene or tag sequence. In a construct, the vector maycomprise one or more recombination sites, one or more stop codons andone or more tag sequences. In some embodiments, the tag sequences may beadjacent to a recombination site. Optionally, a stop codon may beincorporated into the sequence of the tag or in the sequence of therecombination site in order to allow controlled addition of the tagsequence to the gene of interest. In embodiments of this type, the geneof interest may be inserted into the vector by recombinational cloningsuch that the tag and the coding sequence of the gene of interest are inthe same reading frame. The gene of interest may be provided withtranslation initiation signals, e.g., Shine-Delgarno sequences, Kozaksequences and/or IRES sequences, in order to permit the expression ofthe gene with a native N-terminal when the stop codon is not suppressed.The gene of interest may also be provided with a stop codon at the3′-end of the coding sequence. In some embodiments, a tag sequence maybe provided at both the N- and C-terminals of the gene of interest.Optionally, the tag sequence at the N-terminal may be provided with astop codon and the gene of interest may be provided with a stop codonand the tag at the C-terminal may be provided with a stop codon. Thestop codons may be the same or different. In some embodiments, the stopcodon of the N-terminal tag is different from the stop codon of the geneof interest. In embodiments of this type, suppressor tRNAs correspondingto one or both of the stop codons may be provided. When both areprovided, each of the suppressor tRNAs may independently be provided onthe same vector, a different vector or in the host cell genome. Thesuppressor tRNAs need not both be provided in the same way, for example,one may be provided on the vector containing the gene of interest whilethe other may be provided in the host cell genome. In this way, thenucleic acid molecules of one such aspect of the invention may comprisea suppressible stop codon that separates two coding regions. Dependingon the location of the expression signals (e.g., promoters), expressionof the suppressor tRNA results in suppression of the stop codon(s),thereby allowing the production of a fusion peptide, for example afusion peptide having an affinity tag sequence at the N- and/orC-terminus of the expressed protein. By not suppressing the stopcodon(s), expression of the sequence of interest without the N- and/orC-terminal tag sequence may be accomplished. Thus, the invention allowsthrough recombination efficient construction of vectors containing agene or sequence of interest (e.g., one or more open reading frames or“orfs”) for controlled expression of fusion proteins depending on theneed. Preferably, the starting nucleic acid molecules or productmolecules of the invention which are cloned into one or more vectorscomprise at least one open reading frame (orf). Such starting or productmolecules may also comprise functional sequences (e.g., primer sites,transcriptional or translation sites or signals, termination sites(e.g., stop codons which may be optionally suppressed), origins ofreplication, and the like) and preferably comprises sequences thatregulate gene expression including transcriptional regulatory sequencesand sequences that function as internal ribosome entry sites (IRES).Preferably, at least one of the starting or product molecules and/orvectors comprise sequences that function as a promoter. Such starting orproduct molecules and/or vectors may also comprise transcriptiontermination sequences, selectable markers, restriction enzymerecognition sites, and the like.

In some embodiments, the vector comprises two copies of the sameselectable marker, each copy flanked by recombination sites and/ortopoisomerase recognition sites. In other embodiments, the vectorcomprises two different selectable markers each flanked by tworecombination sites. In some embodiments, one or more of the selectablemarkers may be a negative selectable marker.

In a specific aspect, the invention provides a method of cloningcomprising providing at least a first nucleic acid molecule comprisingat least a first and a second recombination site and at least a secondnucleic acid molecule comprising at least a third and a fourthrecombination site, wherein either the first or the second recombinationsite is capable of recombining with either the third or the fourthrecombination site and conducting a recombination reaction such that thetwo nucleic acid molecules are recombined into one or more productnucleic acid molecules and cloning the product nucleic acid moleculesinto one or more vectors. In certain such embodiments, the recombinationsites flank the first and/or second nucleic acid molecules. Moreover,the cloning step is often accomplished by the recombination reaction ofthe product molecule into a vector comprising one or more recombinationsites. In one aspect, the cloning step comprises conducting arecombination reaction between the sites in the product nucleic acidmolecule that did not react in the first recombination reaction with avector having recombination sites capable of recombining with theunreacted sites.

In some embodiments, a recombination site and/or a topoisomeraserecognition site may be attached to a molecule of interest usingconventional conjugation technology. For example, oligonucleotidescomprising the recombination site and/or topoisomerase recognition sitecan be synthesized so as to include one or more reactive functionalmoieties which may be the same or different. Suitable reactivefunctional moieties include, but are not limited to, amine groups, epoxygroups, vinyl groups, thiol groups and the like. The synthesis ofoligonucleotides comprising one or more reactive functional moieties isroutine in the art. Once synthesized, oligonucleotides comprising one ormore reactive functional moieties may be attached to one or morereactive groups present on the molecule or compound of interest. Theoligonucleotides may be attached directly by reacting one or more of thereactive functional moieties with one or more of the reactive functionalgroups. In some embodiments, the attachment may be effected using asuitable linking group capable of reacting with one or more of thereactive functional moieties present on the oligonucleotide and with oneor more of the reactive groups present on the molecule of interest. Inother embodiments, both direct attachment and attachment through alinking group may be used. Those skilled in the art will appreciate thatthe reactive functional moieties on the oligonucleotide may be the sameor different as the reactive functional moieties on the molecules and/orcompounds of interest. Suitable reagents and techniques for conjugationof the oligonucleotide to the molecule of interest may be found inHermanson, Bioconjugate Techniques, Academic Press Inc., San Diego,Calif., 1996.

The invention also relates to compositions for carrying out the methodsof the invention, and kits comprising such compositions, and tocompositions created while carrying out the methods of the invention.

Compositions, methods and kits of the invention may be prepared andcarried out using a phage-lambda site-specific recombination system.Further, such compositions, methods and kits may be prepared and carriedout using the GATEWAY™ Recombinational Cloning System and/or the TOPO®Cloning System and/or the pENTR Directional TOPO® Cloning System, whichare available from Invitrogen Corporation (Carlsbad, Calif.).

In other aspects, the invention provides isolated nucleic acid moleculescomprising one or more (e.g., one, two, three, four, five, etc.)recombination sites and/or one or more (e.g., one, two, three, four,five, etc.) topoisomerase recognition sites. One such molecule of theinvention will contain two or more recombination sites flanking onetopoisomerase recognition site. Another such molecule of the inventionwill contain two or more recombination sites and two or moretopoisomerase recognition sites, wherein each recombination site mayflank a topoisomerase recognition site. Nucleic acid molecules accordingto this aspect of the invention may be linear, circular, or have any ofa variety of geometries and structures, such as coiled, supercoiled,etc. Recombination sites advantageously used in nucleic acid moleculesaccording to this aspect of the invention include, but are not limitedto, att sites (including, but not limited to, attB sites, attP sites,attL sites, attR sites, and the like), lox sites (including, but notlimited to, loxP sites, loxP511 sites, and the like), psi sites, difsites, cer sites, frt sites, and mutants, variants, and derivatives ofthese recombination sites that retain the ability to undergorecombination. Topoisomerase recognition sites advantageously used inthe nucleic acid molecules of this aspect of the invention arepreferably recognized and bound by a type I topoisomerase (such as typeIA topoisomerases (including but not limited to E. coli topoisomerase I,E. coli topoisomerase III, eukaryotic topoisomerase II, archeal reversegyrase, yeast topoisomerase III, Drosophila topoisomerase III, humantopoisomerase III, Streptococcus pneumoniae topoisomerase III, and thetraE protein of plasmid RP4) and type IB topoisomerases (including butnot limited to eukaryotic nuclear type I topoisomerase and a poxvirus(such as that isolated from or produced by vaccinia virus, Shope fibromavirus, ORF virus, fowlpox virus, molluscum contagiosum virus and Amsactamoorei entomopoxvirus)), and type II topoisomerase (including, but notlimited to, bacterial gyrase, bacterial DNA topoisomerase IV, eukaryoticDNA topoisomerase II (such as calf thymus type II topoisomerase), andT-even phage-encoded DNA topoisomerase).

The invention also provides vectors (which may be expression vectors)comprising such isolated nucleic acid molecules. Exemplary vectorsaccording to this aspect of the invention include, but are not limitedto, pcDNAGW-DT(sc), pENTR-DT(sc), pcDNA-DEST41, pENTR/D-TOPO,pENTR/SD/D-TOPO, pcDNA3.2N5/GWD-TOPO and pcDNA6.2N5/GWD-TOPO. Theinvention also provides host cells comprising such the isolated nucleicacid molecules or vectors of the invention.

In related aspects, the invention provides in vitro methods of cloning anucleic acid molecule. Methods according to this aspect of the inventionmay comprise one or more steps, including:

(a) obtaining a nucleic acid molecule to be cloned (which in certainembodiments may be a linear molecule (and which may be blunt-ended ornot) such as a PCR product, and which may optionally comprise one ormore genes or open reading frames);

(b) mixing the nucleic acid molecule to be cloned in vitro with a vector(which may be an expression vector) comprising at least a firsttopoisomerase recognition site flanked by at least a first recombinationsite and at least a second recombination site, wherein the first andsecond recombination sites do not recombine with each other, and with atleast one topoisomerase; and

(c) incubating the mixture under conditions such that the nucleic acidmolecule to be cloned is inserted into the vector between the first andsecond topoisomerase recognition sites, thereby producing a firstproduct molecule comprising the nucleic acid molecule localized betweenthe first and second recombination sites. The invention further includesnucleic acid molecules prepared by the above methods.

Methods according to this aspect of the invention may comprise one ormore additional steps, including, for example, contacting the firstproduct molecule with at least one vector comprising at least a thirdand fourth recombination sites that do not recombine with each other,under conditions favoring recombination between the first and third andbetween the second and fourth recombination sites, thereby producing atleast one second product molecule. According to the invention, the firstand/or second product molecules produced by these methods may beinserted into a host cell. The vectors used in this aspect of theinvention may comprise at least one additional nucleic acid sequenceselected from the group consisting of a selectable marker, a cloningsite, a restriction site, a promoter, an operon, an origin ofreplication, and a gene or partial gene (i.e., a gene fragment orelement).

Recombination sites and topoisomerase recognition sites used in themethods of this aspect of the invention include, but are not limited to,those described elsewhere herein. In particular methods, the secondproduct nucleic acid molecule and the vector are combined in thepresence of at least one recombination protein, which may be but is notlimited to Cre, Int, IHF, Xis, Fis, Hin, Gin, Cin, Tn3 resolvase, TndX,XerC, or XerD. In certain such embodiments, the recombination protein isCre, Int, Xis, IHF or Fis.

The invention also provides kits comprising these isolated nucleic acidmolecules of the invention, which may optionally comprise one or moreadditional components selected from the group consisting of one or moretopoisomerases, one or more recombination proteins, one or more vectors,one or more polypeptides having polymerase activity, and one or morehost cells.

Other preferred embodiments of the invention will be apparent to one orordinary skill in the art in light of what is known in the art, in lightof the following drawings and description of the invention, and in lightof the claims.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 is a schematic representation of a basic recombinational cloningreaction.

FIG. 2 is a schematic representation of the use of the present inventionto clone two nucleic acid segments by performing an LR recombinationreaction.

FIG. 3 is a schematic representation of the use of the present inventionto clone two nucleic acid segments by joining the segments using an LRreaction and then inserting the joined fragments into a DestinationVector using a BP recombination reaction.

FIG. 4 is a schematic representation of the use of the present inventionto clone two nucleic acid segments by performing a BP reaction followedby an LR reaction.

FIG. 5 is a schematic representation of two nucleic acid segments havingattB sites being cloned by performing a first BP reaction to generate anattL site on one segment and an attR on the other followed by an LRreaction to combine the segments. In variations of this process, P1, P2,and/or P3 can be oligonucleotides or linear stretches of nucleotides.

FIG. 6 is a schematic representation of the cloning of two nucleic acidsegments into two separate sites in a Destination Vector using an LRreaction.

FIG. 7 is a schematic representation of the cloning of two nucleic acidsegments into two separate sites in a Destination Vector using a BPreaction.

FIGS. 8A and 8B depict generating a covalently linked double strandednucleotide sequence containing an element on each end according to amethod of the invention. “PCR” indicates polymerase chain reaction;“TOPO” indicates topoisomerase; topoisomerase shown as circle attachedto sequence; “P1” and “P2” indicate PCR primers. Topoisomeraserecognition site is indicated in bold print. (5′-CGGAACAAGGG (SEQ ID NO:63); 3′-GGGAACCGGAT (SEQ ID NO: 64); 5′-CCCTTCGGAACAAGGG (SEQ ID NO:65); 5′-CCCTTGGCCATAAGGG (SEQ ID NO: 66); 5′-GGCCATAAGGG (SEQ ID NO:135); 3′-GGGAAGCCTTG (SEO ID NO: 136))

FIGS. 9A-9D show the ends of PCR products representing a cytomegaloviruspromoter element (“CMV”), a green fluorescent protein element (“GFP”),and a bovine growth hormone polyadenylation signal (“BGH”) element.Primers used to construct the PCR products of FIGS. 9A, 9B and 9C areindicated by an “F” number (see FIG. 9D). The portion of one or bothends including the topoisomerase recognition site (CCCTT) is shown. Boldprint indicates overhanging sequences. In FIGS. 9A and 9B, one (FIG. 9B)or both (FIG. 9A) of the overhang sequences are palindromic in nature.Sequences are shown in conventional orientation, with the top strand ina 5′ to 3′ orientation from left to right, and the bottom strand in a 3′to 5′ orientation from left to right. Number in parentheses above orbelow sequences indicates SEQ ID NOs.

FIGS. 10A and 10B show constructs (FIG. 10A) and results (FIG. 10B) ofstudies examining the ability to use covalently linked ds recombinantnucleic acid molecules that encode polypeptides for performing a twohybrid assay. FIG. 10A shows the amount of each construct used fortransfection. A “p” preceding an amount or volume of reactant indicatesplasmid form, “1” indicates linear form, and “PCR” indicates PCRamplification reaction mixture. FIG. 10B shows the level ofβ-galactosidase activity (“LacZ activity”) associated with eachtransfected sample. Increased LacZ activity is indicative of a positiveinteraction.

FIGS. 11A to 11F represent various embodiments of the composition andmethods for generating a ds recombinant nucleic acid molecule covalentlylinked in one strand. Note nicks in one or both strands of the moleculesshown in FIGS. 11B-11F.

FIGS. 12A to 12D illustrate various embodiments of compositions andmethods of the invention for generating a covalently linked dsrecombinant nucleic acid molecule. Topoisomerase is shown as a solidcircle, and is either attached to a terminus of a substrate nucleic acidmolecule or is released following a linking reaction. As illustrated,the substrate nucleic acid molecules have 5′ overhangs, although theysimilarly can have 3′ overhangs or can be blunt ended. In addition,while the illustrated nucleic acid molecules are shown having thetopoisomerases bound thereto (topoisomerase-charged), one or more of thetermini shown as having a topoisomerase bound thereto also can berepresented as having a topoisomerase recognition site, in which casethe joining reaction would further require addition of one or more sitespecific topoisomerases, as appropriate.

FIG. 12A shows a first nucleic acid molecule having a topoisomeraselinked to each of the 5′ terminus and 3′ terminus of one end, andfurther shows linkage of the first nucleic acid molecule to a secondnucleic acid molecule.

FIG. 12B shows a first nucleic acid molecule having a topoisomerasebound to the 3′ terminus of one end, and a second nucleic acid moleculehaving a topoisomerase bound to the 3′ terminus of one end, and furthershows a covalently linked ds recombinant nucleic acid molecule generateddue to contacting the ends containing the topoisomerase-chargedsubstrate nucleic acid molecules.

FIG. 12C shows a first nucleic acid molecule having a topoisomerasebound to the 5′ terminus of one end, and a second nucleic acid moleculehaving a topoisomerase bound to the 5′ terminus of one end, and furthershows a covalently linked ds recombinant nucleic acid molecule generateddue to contacting the ends containing the topoisomerase-chargedsubstrate nucleic acid molecules.

FIG. 12D shows a nucleic acid molecule having a topoisomerase linked toeach of the 5′ terminus and 3′ terminus of both ends, and further showslinkage of the topoisomerase-charged nucleic acid molecule to twonucleic acid molecules, one at each end. The topoisomerases at each ofthe 5′ termini and/or at each of the 3′ termini can be the same ordifferent.

FIG. 13 illustrates the generation of an expressible ds recombinantnucleic acid molecule and amplification of the expressible dsrecombinant nucleic acid molecule. The expressible ds recombinantnucleic acid molecule is generated from three nucleic acid molecules,including a nucleotide sequence comprising a promoter, a nucleotidesequence comprising a coding sequence, and a nucleotide sequencecomprising a polyadenylation signal. Generation of the nucleic acidmolecule can be facilitated by the incorporation of complementary 5′and/or 3′ overhanging sequences at the ends of the ds nucleotidessequences to be joined. The expressible ds recombinant nucleic acidmolecule is generated by contacting a first nucleic acid molecule havinga type IA topoisomerase at a 5′ terminus of a first end and a type IBtopoisomerase at a 3′ terminus of a second end, with a second nucleicacid molecule and a third double stranded nucleotide sequence. Theexpressible ds recombinant nucleic acid molecule is amplified using afirst primer that hybridizes to the second ds recombinant nucleic acidmolecule upstream of the promoter, and a second primer that hybridizesto the third ds recombinant nucleic acid molecule downstream of thepolyadenylation signal.

FIG. 14 shows one example of a process for preparing a double strandednucleic acid molecule which contains a topoisomerase (e.g., a type IAtopoisomerase) bound to the 5′ terminus of one end of the molecule,wherein the same end of the molecule further comprise a 3′ overhang (see(4) in this figure).

FIG. 15 shows two embodiments of the invention in which a singlestranded or double stranded DNA nucleotide sequence is joined withsingle stranded RNA nucleotide sequence.

FIG. 16 is a schematic demonstrating the flexibility in entry point forPCR cloning using the TOPO-Gateway™ or standard Gateway™ cloningmethodologies.

FIG. 17 is a schematic diagram of the production of expression clonesusing the Gateway™ system and a directional TOPO-Gateway™ expressionvector.

FIG. 18 is a map of the multiple cloning site in plasmids pcDNAGW-DT(sc)and pENTR-DT(sc). (SEQ ID NO: 67; amino acid sequences SEQ ID NO: 68 andSEQ ID NO: 69)

FIG. 19 is a physical map of plasmid pcDNAGW-DT.

FIG. 20 is a physical map of plasmid pcDNA-DEST41.

FIG. 21 is a physical map of plasmid pENTR-DT.

FIGS. 22A-22B are depictions of the physical map (FIG. 22A) showing theTOPO cloning site in, and the nucleotide sequence (FIG. 22B (SEQ ID NO.70) of, plasmid pENTR/D-TOPO. The physical map depicts the adapted,supercoiled form of the vector, while the nucleotide sequence depictsthe vector containing a start codon and an open reading frame (atgnnnnnn. . . ). Restriction sites are labeled to indicate the actual cleavagesite. The boxed region indicates attL sequences in the entry clone thatwill be transferred into the destination vector following recombination.

FIGS. 23A-23B are depictions of the physical map (FIG. 23A) showing theTOPO cloning site in, and the nucleotide sequence (FIG. 23B (SEQ ID NO.71)) of, plasmid pENTR/SD/D-TOPO. The physical map depicts the adapted,supercoiled form of the vector, while the nucleotide sequence depictsthe vector containing a start codon and an open reading frame (atgnnnnnn. . . ). Restriction sites are labeled to indicate the actual cleavagesite. The boxed region indicates attL sequences in the entry clone thatwill be transferred into the destination vector following recombination.

FIGS. 24A-24C are depictions of the physical map (FIG. 24A) and thenucleotide sequence (FIG. 24B-C) (SEQ ID NO: 72) of plasmidpcDNA3.2N5/GWD-TOP07. The physical map depicts the adapted, supercoiledform of the vector, while the nucleotide sequence depicts the vectorcontaining a start codon and an open reading frame (atgnnnnnn . . . ).

FIGS. 25A to 25C are depictions of a physical map (FIG. 25A) and thenucleotide sequence (FIG. 25B-C) (SEQ ID NO: 73) of plasmidpcDNA6.2N5/GWD-TOP07. The physical map depicts the adapted, supercoiledform of the vector, while the nucleotide sequence depicts the vectorcontaining a start codon and an open reading frame (atgnnnnnn . . . ).

FIG. 26 is a depiction of an exemplary adaptation strategy forpENTR/SD-dTopo (SEQ ID NO: 74: SEQ ID NO: 75: SEQ ID NO: 138: SEQ ID NO139), pENTR-dTopo (SEQ ID NO: 76: SEQ ID NO: 78: SEQ ID NO: 140: SEQ IDNO: 141), and pcDNAGW-dTopo (SEQ ID NO: 77: SEQ ID NO: 79: SEQ ID NO:142).

FIG. 27 is a photograph of a Western blot analysis of HLA and CATexpressed in COS cells. The genes encoding CAT (26 kDa) and HLA (41 kDa)were amplified by PCR and either Topo-cloned into pENTR-dTopo andtransferred into pcDNA-DEST40 (lanes 2 and 5, respectively), or cloneddirectly into pcDNAGW-dTopo (lanes 3 and 6, respectively). Theseconstructs were used to transfect COS cells and the lysates probed forrecombinant V5 tagged protein by Western blot, using V5-HRP antibodyconjugate. Lanes 1 and 4 represent cells only controls.

FIG. 28 is a photograph of a gel depicting HLA and CAT expression in E.coli. The genes encoding HLA (41 kDa) and CAT (26 kDa) were amplified byPCR and either topo cloned into pENTR/SD-dTopo and transferred intopET-DEST42 (lanes 3 and 6, respectively) or cloned directly intopET101-dTopo (lanes 4 and 7, respectively). These constructs were usedto transform BL21(DE3) cells and induced to express by addition of IPTGto 1 mM for 3 hours at 37 C. Cell lysates were run on a NuPage andstained with SafeStain™. Lanes 2 and 5 represent cells uninduced celllysates from the respective pET-DEST42 cultures.

FIG. 29 is a schematic depiction of the binding of a topoisomerase to arecognition site near the 3′ terminus of a target nucleic acid molecule.Upon binding of the topoisomerase, the downstream sequence (3′ to thecleavage site) can dissociate, leaving a nucleic acid molecule havingthe topoisomerase covalently bound to the newly generated 3′ end. (SEQID NO: 80)

FIGS. 30A-30C are depictions protein expression results (Western blot)for mammalian expression cassettes that were constructed by PCRamplification of expression elements and a gene of interest (CAT or V5)followed by a TOPO joining reaction performed with or without secondaryPCR. Protein expression data from the expression cassette transfectedinto suspension TRex-CHO cells (FIG. 30A), adherent TRex-CHO cells (FIG.30B), and adherent TRex-293 cells (FIG. 30C). For the Western blot,anti-V5 or anti-CAT antibodies were used for detection. Arrows indicatethe position of the bands corresponding to the V5 or CAT proteins.

FIG. 31 is a photograph of an ethidium bromide-stained agarose gelcontaining PCR products showing that the Gateway-compatible cassettecontained inserts of the expected size. The Gateway-compatible cassettewas constructed by first generating a CAT insert by PCR and then using aTOPO joining reaction to introduce attB1 and attB2 adaptors. Thepurified DNA product was inserted into pDONR 222 using a BP reaction.Following transformation into E. coli, PCR was performed on the coloniesand the PCR product was checked on an ethidium bromide-stained agarosegel.

FIG. 32 is a schematic diagram depicting the preparation oftopoisomerase-charged pENTR vectors, by charging pDONR vectors withtopoisomerase and carrying out a BxP GATEWAY cloning reaction accordingto methods of the invention.

FIG. 33 is a schematic diagram depicting the preparation oftopoisomerase-charged pEXP vectors, by charging pDEST vectors withtopoisomerase and carrying out an LxR GATEWAY cloning reaction, thenadding TOPO adaptors to the cut ends of the pEXP vector, according tomethods of the invention.

FIG. 34 shows a schematic outline of methods of the invention. In thefirst step, nucleic acid molecules to be assembled are generated using,for example, PCR. In the second step, nucleic acid molecules of thefirst step are assembled using methods of the invention (e.g., methodsinvolving the use of topoisomerase to covalently linking at least onestrand of one nucleic acid segments to another nucleic acid segment). Inthe third step, assembled nucleic acid molecules generated in the secondstep either may be used directly or may be amplified and then used.Examples of uses of the assembled molecules are described elsewhereherein.

FIG. 35 shows a schematic representation of a process for usingtopoisomerase to link two nucleic acid segments, followed by single siterecombination to recombine the linked nucleic acid segment with anothernucleic acid segment. In the first step, a topoisomerase adapted nucleicacid segment which contains an attL1 recombination site is linked toanother nucleic acid segment, referred to here as an insert (labeled“I”), using any of the topoisomerase mediated methods described hereinfor connecting nucleic acid molecules. The topoisomerase assemblednucleic acid segments are then contacted with another nucleic acidsegment which contains a promoter, labeled “P”, and an attR1recombination site in the presence of LR CLONASE™ under conditions whichallow for recombination between the two recombination sites.Recombination results in the formation of a nucleic acid molecule whichcontains the insert nucleic acid segment in operable linkage with thepromoter. Further, an attB1 recombination site is located between thepromoter and the insert in the end product. The recombination sitesshown in this figure are attL and attB sites, but any suitablerecombination sites could be used.

FIG. 36 shows a schematic representation of a process for usingtopoisomerase and recombination to recombine and/or link five separatenucleic acid segments and circularize the resulting product. In thefirst step, a topoisomerase adapted nucleic acid segment which containsattL 1 and attL2 recombination sites and a negative selection marker(labeled “NM”) is linked to another nucleic acid segment, referred tohere as an insert (labeled “I”), using any of the topoisomerase mediatedmethods described herein for connecting nucleic acid molecules. Thetopoisomerase assembled nucleic acid segments are then contacted withtwo additional nucleic acid segments, each of which contains at leastone attR recombination site, in the presence of LR CLONASE™ (InvitrogenCorporation, Carlsbad, Calif.) under conditions which allow forrecombination between the various recombination sites. In certain suchmethods, for example, TOPO-adapted vectors are incubated with one ormore nucleic acid segments (e.g., one or more PCR products) at roomtemperature (e.g., about 20-20° C.) for about 5-30 (and preferably about10) minutes; the reaction is then heat-treated by incubation at about80° C. for about 20 minutes, and the reaction mixture then used in astandard LR reaction according to manufacturer's instructions(Invitrogen Corporation, Carlsbad Calif.), except the incubation timefor the LR reaction is increased to about 3 hours. The recombinationreactions result in the formation of a product molecule in which thepromoter is linked to (1) the insert molecule and (2) an origin ofreplication (labeled “ori”). This product molecule is then connected toa nucleic acid segment which is topoisomerase adapted at both terminiand contains a positive selection marker (labeled “PM”). Further, thefinal topoisomerase linkage step results in the formation of a circularnucleic acid molecule. The recombination sites shown in this figure areattL and attB sites, but any suitable recombination sites could be used.

FIG. 37 shows a schematic representation of a process for thepreparation of nucleic acid molecules for performing homologousrecombination. In this instance, three nucleic acid segments areconnected to each other using methods which involve topoisomerasemediated covalent linkage of nucleic acid strands of the individualsegments. Two of these nucleic acid segments each contain a positiveselection marker and two attL sites which flank a negative selectionmarker. Thus, the nucleic acid molecule which results from the firststep contains a nucleic acid segment, referred to here as an insert. Oneach side of the insert is (1) a positive selection marker and (2) tworecombination sites which flank a negative selection marker. LR CLONASE™catalyzed recombination in the presence of two nucleic acid segmentswhich contain regions that share homology to a chromosomal locus wherethe nucleic acid end product is designed to integrate (labeled “HR1” and“HR2”) results in the formation of the end product nucleic acid moleculeshown. As one skilled in the art would recognize, any suitablerecombination sites could be used in the process set out in this figure.

FIG. 38 shows a schematic representation of the linking of four nucleicacid segments using toposiomerase to generate a linear nucleic acidmolecule with recombination sites (labeled “L1” and “L2”) located nearthe termini. Upon toposiomerase mediated linkage of the nucleic acidstrands, no nicks are present at the junction points. In a second step,the topoisomerase assembled nucleic acid segments are contacted withanother nucleic acid segment which contains an origin of replication(labeled “ori”), a positive selection marker (labeled “PM”), an attR1recombination site, and an attR2 recombination site in the presence ofLR CLONASE™ under conditions which allow for recombination between therecombination sites. Recombination results in the formation of acircular nucleic acid molecule as shown. The recombination sites shownin this figure are attL and attB sites, but any suitable recombinationsites could be used.

FIG. 39 shows a schematic representation of the linking of two nucleicacid segments in a single step process using toposiomerase andrecombination sites to generate a circular nucleic acid molecule. One ofthe nucleic acid segments contains an attL1 recombination site (labeled“L1”), a promoter (labeled “P”), and toposiomerase molecule covalentlylinked to one terminus. The other nucleic acid segment contains an attR1recombination site (labeled “R1”), an open reading frame (labeled“ORF”), an origin of replication (labeled “ORI”), a positive selectionmarker (labeled “PM”), and topoisomerase molecule covalently linked toone terminus. Thus, when these two nucleic acid segments are contactedwith each other in the presence of LR CLONASE™ under conditions whichallow for recombination between the attL and attR recombination sitesand topoisomerase mediated linkage of nucleic acid strands, a circularmolecule is formed having the structure indicated. The recombinationsites shown in this figure are attL and attB sites, but any suitablerecombination sites could be used.

FIG. 40 shows a schematic representation of the linking of two nucleicacid segments using toposiomerase mediated methods to generate acircular nucleic acid molecule. This circular molecule contains an openreading frame (labeled “ORF”) positioned between attL1 and attL2recombination site (labeled “L1” and “L2”). The topoisomerase assembledproduct then undergoes recombination with another circular moleculewhich contains attR1 and attR2 recombination sites to generate a thirdcircular nucleic acid molecule which contains the open reading framepositioned between attB1 and attB2 recombination sites. Further, theopen reading frame is operably linked to a promoter. The recombinationsites shown in this figure are attL and attB sites, but any suitablerecombination sites could be used.

FIG. 41 shows an example of a process by which two nucleic acid segmentsmay be covalently linked to each other in both strands at the junctionwhere the two nucleic acid segments are connected. As in other figurespresented herein, the “lollipop” type symbol represents a topoisomerasemolecule. Further, the arrows within the boxes represent the functionaldirectionality of the particular nucleic acid segment. For example, ifthe PCR product is an open reading frame, then the 3′ end of the codingregion (i.e., the end of the coding region which encodes the C-terminalend of the polypeptide) would be at the point of the arrow and the 5′end would be at the other end.

FIGS. 42A-42B. T7 TOPO-linker. (A) Diagram of TOPO-activated T7 linker.Three oligonucleotides were annealed, incubated with TOPO, and thediagrammed TOPO-DNA covalent complex was purified as described inMaterials and Methods. (see FIG. 42A) The T7 promoter is shown in bold,and the TOPO recognition site is underlined. (B) Use of TOPO-linker toadd a T7 RNA polymerase promoter to a PCR product generated by Tagpolymerase (A-tailed) (see FIG. 42B).

FIGS. 43A-43B. EPLC purification. (A) Chromatogram of FPLC purificationof TOPO-activated T7 linker. A₂₅₄ trace, conductivity (saltconcentration), and the relative concentration of a buffer B in themixing chamber. The boundaries of eleven 1 ml flow-through fractions and39-0.2 ml elution fractions are designated with broken red lines. TheA₂₅₄ trace is actually shifted approximately 3 elution fractions to theleft as determined by gel analysis (see FIG. 43A). (B) Magnified A₂₅₄peak showing small right shoulder corresponding to the position of freetopoisomerase (see also FIG. 43B).

FIGS. 44A-44B. Gel analysis of analysis of fractions. (A) EtBr-stained10% TBE polyacrylamide gels. Except the undigested load, all fractionswere incubated with proteinase K prior to loading. 10 bp DNA ladder,annealed oligos, the load, flow-through (F-T) fractions 2-5, and elutionfractions 29-40 are shown. (see FIG. A) (B) Coomassie-stained 4-12%Bis-Tris NuPAGE gels. The sizes of some of the marker bands in kDA areshown. “TOPO” designates the free topoisomerase control lane (seeMaterials & Methods). (see FIG. B) Load, flow-through, and elutionfractions are labeled as in (A).

FIGS. 45A-45D. PCR, linking reactions, and transcription. (FIGS. A toD). EtBr-stained 1.2% argose-TAE gels. (FIGS. A and C) The 1.2 kb ladderband corresponds to 25 ng DNA. (A) Primary PCR reactions loaded toestimate product yields. “T7-” indicates that the reverse primercontained a 5′ T7 promoter sequence. (see FIG. A) (B) The products oflinking reactions with (“+”) or without (“−”) T7 TOPO linker and withactin or GFP primary PCR products from (A). (see FIG. B) (C) Productsfrom the secondary amplification of the linking reactions in (B). Thevolume loaded from each PCR reaction is indicated. “F+T” indicates thatgene-specific forward primers and T7amp1 primer were used in theamplification. “T” indicates that the T7amp1 primer alone was used.Negative control (“neg ctrl”) reactions used the mock linking reactionsin (B) as templates. (see FIG. C) (D) Products of transcription. “negctrl” transcriptions used the negative control “F+T” secondaryamplification products shown in (C). The “T7-” transcriptions wereperformed with the “T7-” primary PCR products in A as templates (seeFIG. D).

FIGS. 46A-46D. pUC19/actin positive control plasmid. (A) Vector map ofthe positive control plasmid. The actin template sequence was clonedinto BamHI and HindIII sites in the pUC19 polylinker as described inMaterials & Methods. (B)-(D) Photographs of ethidium bromide stainedgels. (see FIG. A) (B) 6% polyacrylamide TBE gels of linking reactionsof a primary PCR product amplification from pUC19/actin with actinF andactinR primers in the presence (right) or absence (left) or T7 TOPOlinker. (see FIG. B) (C) 1.2% agarose-TAE gel of secondary amplificationproduct of linked actin from (B) and the primary product of pUC19/actinamplified with actinF and T7-actinR primers. (see FIG. C) (D) 1.2%agarose-TAE gel DNase I digested transcription reactions using the PCRproducts in (C) as templates (see FIG. D).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms used in recombinantnucleic acid technology are utilized extensively. In order to provide aclear and more consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

Gene: As used herein, a gene is a nucleic acid sequence that containsinformation necessary for expression of a polypeptide, protein orfunctional RNA (e.g., a ribozyme, tRNA, rRNA, mRNA, etc.). It includesthe promoter and the structural gene open reading flame sequence (orf)as well as other sequences involved in expression of the protein.

Structural gene: As used herein, a structural gene refers to a nucleicacid sequence that is transcribed into messenger RNA that is thentranslated into a sequence of amino acids characteristic of a specificpolypeptide.

Host: As used herein, a host is any prokaryotic or eukaryotic organismthat is a recipient of a replicable expression vector, cloning vector orany nucleic acid molecule. The nucleic acid molecule may contain, but isnot limited to, a structural gene, a transcriptional regulatory sequence(such as a promoter, enhancer, repressor, and the like) and/or an originof replication. As used herein, the terms “host,” “host cell,”“recombinant host” and “recombinant host cell” may be usedinterchangeably. For examples of such hosts, see Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982).

Derivative: As used herein, a “derivative” of a specified host is aprogeny of the specified host, a modified or mutated host obtained orderived from the specified host or its progeny, or other recipient hostthat contains genetic material obtained directly or indirectly from thespecified host. Such a derivative host may, for example, be formed byremoving genetic material from a specified host and subsequentlyintroducing it into another host (i.e., the progeny or other recipienthost) by any conventional methodology including, but not limited to,transformation, conjugation, electroporation, transduction and the like.A derivative may be formed by introducing one or more mutations ormodifications into the genome or other genetic material (e.g. vectors,plasmids, extrachromosomal elements, etc.) of a host. Such mutations ormodifications may include one or more insertion mutations, deletionmutations and/or substitutions or various combinations thereof. Themutations or modifications may be insertions into the genome or othergenetic material (e.g. vectors, plasmids, extrachromosomal elements,etc.) of the host. Alternatively, the mutations may be deletions of oneor more bases and/or nucleic acid sequences from the genome or othergenetic material (e.g. vectors, plasmids, extrachromosomal elements,etc.) of the host. In some instances, the mutations may be thealteration of one or more bases in the genome of the host. Suchmodifications or mutations may also comprise substituting one or morenucleic acid bases and/or nucleic acid molecules for other nucleic acidmolecules and/or bases. In addition, one host is a derivative of aparent host if it contains the genome of the parent host but does notcontain some or all of the same extrachromosomal nucleic acid molecules.Thus, a strain produced by curing some or all of the endogenous vectorsfrom a parent strain is a derivative of the parent strain. Derivativesof a host of the invention may also include those hosts obtained by theaddition of one or more nucleic acid molecules into the host ofinterest. Nucleic acid molecules which may be introduced into a hostwill be recognized by one skilled in the art and may include, but is notlimited to, vectors, plasmids, transposons, oligonucleotides, RNA, DNA,RNA/DNA hybrids, phage sequences, virus sequences, regardless of theform or conformation (e.g. linear, circular, supercoiled, singlestranded, double stranded, single/double stranded hybrids and the like).Examples of mutations or other genetic alterations which may beincorporated into the hosts of the present invention include, but arenot limited to, mutations or alterations that create: a recK genotypesuch as recA1/recA13 or recA deletions, a lacZ-genotype that allowsalpha complementation such as lacX74, lacZΔM15 or other lacZ deletion, aprotease deficient genotype such as Δlon and/or ompT⁻, an endonucleaseminus genotype such as endA1, a genotype suitable for M13 phageinfection by including the F′ episome, a restriction negative,modification positive genotype such as hsdR17(r_(K) ⁻, m_(K) ⁺), arestriction negative, modification negative genotype such ashsdS20(r_(B) ⁻, m_(B) ⁻), a methylase deficient genotype such as mcrAand/or mcrB and/or mrr, a genotype suitable for taking up large plasmidssuch as deoR, a genotype containing suppressor mutations such as supEand/or supF. Other suitable modifications are known to those skilled inthe art and such modifications are considered to be within the scope ofthe present invention.

Transcriptional Regulatory Sequence: As used herein, transcriptionalregulatory sequence is a functional stretch of nucleotides contained ona nucleic acid molecule, in any configuration or geometry, that acts toregulate the transcription of one or more structural genes intomessenger RNA. Examples of transcriptional regulatory sequences include,but are not limited to, promoters, operators, enhancers, repressors, andthe like. Transcriptional regulatory sequences may also regulate thetranscription of nucleic acid molecules which encode functional RNAs(e.g., ribozymes, tRNAs, rRNAs, mRNAs, etc.).

Promoter: As used herein, a promoter is an example of a transcriptionalregulatory sequence, and is specifically a nucleic acid sequencegenerally described as the 5′-region of a gene located proximal to thestart codon. The transcription of an adjacent nucleic acid segment isinitiated at the promoter region. A repressible promoter's rate oftranscription decreases in response to a repressing agent. An induciblepromoter's rate of transcription increases in response to an inducingagent. A constitutive promoter's rate of transcription is notspecifically regulated, though it can vary under the influence ofgeneral metabolic conditions.

Insert: As used herein, an insert is a desire nucleic acid segment thatis a part of a larger nucleic acid molecule.

Target Nucleic Acid Molecule: As used herein, target nucleic acidmolecule is a nucleic acid segment of interest preferably nucleic acidwhich is to be acted upon using the compounds and methods of the presentinvention. Such target nucleic acid molecules preferably contain one ormore genes or portions of genes.

Insert Donor: As used herein, an insert donor is one of the two parentalnucleic acid molecules (e.g. RNA or DNA) of the present invention whichcarries the Insert. The Insert Donor molecule comprises the Insertflanked on both sides with recombination sites. The Insert Donor can belinear or circular. In one embodiment of the invention, the Insert Donoris a circular nucleic acid molecule, optionally supercoiled, and furthercomprises a cloning vector sequence outside of the recombination signals(see FIG. 1). When a population of Inserts or population of nucleic acidsegments are used to make the Insert Donor, a population of InsertDonors result and may be used in accordance with the invention.

Product: As used herein, a product is one the desired daughter moleculescomprising the A and D sequences which is produced after the secondrecombination event during the recombinational cloning process (see FIG.1). The Product contains the nucleic acid which was to be cloned orsubcloned. In accordance with the invention, when a population of InsertDonors are used, the resulting population of Product molecules willcontain all or a portion of the population of Inserts of the InsertDonors and preferably will contain a representative population of theoriginal molecules of the Insert Donors.

Recognition sequence: As used herein, a recognition sequence(alternatively and equivalently referred to herein as a “recognitionsite”) is a particular sequence to which a protein, chemical compound,DNA, or RNA molecule (e.g., restriction endonuclease, a topoisomerase, amodification methylase, or a recombinase) recognizes and binds. In thepresent invention, a recognition sequence will usually refer to arecombination site (which may alternatively be referred to as arecombinase recognition site) or a topoisomerase recognition site. Forexample, the recognition sequence for Cre recombinase is loxP which is a34 base pair sequence comprised of two 13 base pair inverted repeats(serving as the recombinase binding sites) flanking an 8 base pair coresequence. See FIG. 1 of Sauer, B., Current Opinion in Biotechnology5:521-527 (1994). Other examples of such recognition sequences are theattB, attP, attL, and attR sequences which are recognized by therecombinase enzyme (Integrase. attB is an approximately 25 base pairsequence containing two 9 base pair core-type Int binding sites and a 7base pair overlap region. attP is an approximately 240 base pairsequence containing core-type Int binding sites and arm-type Int bindingsites as well as sites for auxiliary proteins integration host factor(IHF), FIS and excisionase (Xis). See Landy, Current Opinion inBiotechnology 3:699-707 (1993). Such sites may also be engineeredaccording to the present invention to enhance production of products inthe methods of the invention. When such engineered sites lack the P1 orH1 domains to make the recombination reactions irreversible (e.g., attRor attP), such sites may be designated attR′ or attP′ to show that thedomains of these sites have been modified in some way. Examples oftopoisomerase recognitions sites include, but are not limited to, thesequence 5′-GCAACTT-3′ that is recognized by E. coli topoisomerase III(a type I topoisomerase); the sequence 5′-(C/T)CCTT-3′ which is atopoisomerase recognition site that is bound specifically by mostpoxvirus topoisomerases, including vaccinia virus DNA topoisomerase I;and others that are known in the art as discussed elsewhere herein.

Recombination proteins: As used herein, recombination proteins includeexcisive or integrative proteins, enzymes, co-factors or associatedproteins that are involved in recombination reactions involving one ormore recombination sites, which may be wild-type proteins (See Landy,Current Opinion in Biotechnology 3:699-707 (1993)), or mutants,derivatives (e.g., fusion proteins containing the recombination proteinsequences or fragments thereof), fragments, and variants thereof

Recombination site: A used herein, a recombination site is a recognitionsequence on a nucleic acid molecule participating in anintegration/recombination reaction by recombination proteins.Recombination sites are discrete sections or segments of nucleic acid onthe participating nucleic acid molecules that are recognized and boundby a site-specific recombination protein during the initial stages ofintegration or recombination. For example, the recombination site forCre recombinase is loxP which is a 34 base pair sequence comprised oftwo 13 base pair inverted repeats (serving as the recombinase bindingsites) flanking an 8 base pair core sequence. See FIG. 1 of Sauer, B.,Curr. Opin. Biotech. 5:521-527 (1994). Other examples of recognitionsequences include the attB, attP, attL, and attR sequences describedherein, and mutants, fragments, variants and derivatives thereof, whichare recognized by the recombination protein (Int and by the auxiliaryproteins integration host factor (IHF), FIS and excisionase (Xis). SeeLandy, Curr. Opin. Biotech. 3:699-707 (1993).

Recombinational Cloning: As used herein, recombinational cloning is amethod, such as that described in U.S. Pat. Nos. 5,888,732, 6,143,557,6,171,861, 6,270,969, and 6,277,608 (the contents of which are fullyincorporated herein by reference), and as also described herein, wherebysegments of nucleic acid molecules or populations of such molecules areexchanged, inserted, replaced, substituted or modified, in vitro or invivo. Preferably, such cloning method is an in vitro method.

Repression cassette: As used herein, repression cassette is a nucleicacid segment that contains a repressor or a Selectable marker present inthe subcloning vector.

Selectable marker: As used herein, selectable marker is a nucleic acidsegment that allows one to select for or against a molecule (e.g., areplicon) or a cell that contains it, often under particular conditions.These markers can encode an activity, such as, but not limited to,production of RNA, peptide, or protein, or can provide a binding sitefor RNA, peptides, proteins, inorganic and organic compounds orcompositions and the like. Examples of selectable markers include butare not limited to: (1) nucleic acid segments that encode products whichprovide resistance against otherwise toxic compounds (e.g.,antibiotics); (2) nucleic acid segments that encode products which areotherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophicmarkers); (3) nucleic acid segments that encode products which suppressthe activity of a gene product; (4) nucleic acid segments that encodeproducts which can be readily identified (e.g., phenotypic markers suchas (-galactosidase, green fluorescent protein (GFP), and cell surfaceproteins); (5) nucleic acid segments that bind products which areotherwise detrimental to cell survival and/or function; (6) nucleic acidsegments that otherwise inhibit the activity of any of the nucleic acidsegments described in Nos. 1-5 above (e.g., antisense oligonucleotides);(7) nucleic acid segments that bind products that modify a substrate(e.g. restriction endonucleases); (8) nucleic acid segments that can beused to isolate or identify a desired molecule (e.g. specific proteinbinding sites); (9) nucleic acid segments that encode a specificnucleotide sequence which can be otherwise non-functional (e.g., for PCRamplification of subpopulations of molecules); (10) nucleic acidsegments, which when absent, directly or indirectly confer resistance orsensitivity to particular compounds; and/or (11) nucleic acid segmentsthat encode products which are toxic in recipient cells.

Selection scheme: As used herein, selection scheme is any method whichallows selection, enrichment, or identification of a desired Product orProduct(s) from a mixture containing an Entry Clone or Vector, aDestination Vector, a Donor Vector, an Expression Clone or Vector, anyintermediates (e.g. a Cointegrate or a replicon), and/or Byproducts. Theselection schemes of one preferred embodiment have at least twocomponents that are either linked or unlinked during recombinationalcloning. One component is a Selectable marker. The other componentcontrols the expression in vitro or in vivo of the Selectable marker, orsurvival of the cell (or the nucleic acid molecule, e.g., a replicon)harboring the plasmid carrying the Selectable marker. Generally, thiscontrolling element will be a repressor or inducer of the Selectablemarker, but other means for controlling expression or activity of theSelectable marker can be used. Whether a repressor or activator is usedwill depend on whether the marker is for a positive or negativeselection, and the exact arrangement of the various nucleic acidsegments, as will be readily apparent to those skilled in the art. Insome preferred embodiments, the selection scheme results in selection ofor enrichment for only one or more desired Products. As defined herein,selecting for a nucleic acid molecule includes (a) selecting orenriching for the presence of the desired nucleic acid molecule, and (b)selecting or enriching against the presence of nucleic acid moleculesthat are not the desired nucleic acid molecule.

In one embodiment, the selection schemes (which can be carried out inreverse) will take one of three forms, which will be discussed in termsof FIG. 1. The first, exemplified herein with a Selectable marker and arepressor therefore, selects for molecules having segment D and lackingsegment C. The second selects against molecules having segment C and formolecules having segment D. Possible embodiments of the second formwould have a nucleic acid segment carrying a gene toxic to cells intowhich the in vitro reaction products are to be introduced. A toxic genecan be a nucleic acid that is expressed as a toxic gene product (a toxicprotein or RNA), or can be toxic in and of itself (In the latter case,the toxic gene is understood to carry its classical definition of“heritable trait”.)

Examples of such toxic gene products are well known in the art, andinclude, but are not limited to, restriction endonucleases (e.g., DpnI),apoptosis-related genes (e.g. ASK1 or members of the bcl-2/ced-9family), retroviral genes including those of the human immunodeficiencyvirus (HIV), defensins such as NP-1, inverted repeats or pairedpalindromic nucleic acid sequences, bacteriophage lytic genes such asthose from (X174 or bacteriophage T4; antibiotic sensitivity genes suchas rpsL, antimicrobial sensitivity genes such as pheS, plasmid killergenes, eukaryotic transcriptional vector genes that produce a geneproduct toxic to bacteria, such as GATA-1, and genes that kill hosts inthe absence of a suppressing function, e.g., kicB, ccdb, (X174 E (Liu,Q. et al., Curr. Biol. 8:1300-1309 (1998)), and other genes thatnegatively affect replicon stability and/or replication. A toxic genecan alternatively be selectable in vitro, e.g., a restriction site.

Many genes coding for restriction endonucleases operably linked toinducible promoters are known, and may be used in the present invention.See, e.g. U.S. Pat. No. 4,960,707 (DpnI and DpnII); U.S. Pat. Nos.5,000,333, 5,082,784 and 5,192,675 (KpnI); U.S. Pat. No. 5,147,800(NgoARIII and NgoAI); U.S. Pat. No. 5,179,015 (FspI and HaeIII): U.S.Pat. No. 5,200,333 (HaeII and TaqI); U.S. Pat. No. 5,248,605 (HpaII);U.S. Pat. No. 5,312,746 ClaI); U.S. Pat. Nos. 5,231,021 and 5,304,480(XhoI and XhoII); U.S. Pat. No. 5,334,526 (AluI); U.S. Pat. No.5,470,740 (NsiI); U.S. Pat. No. 5,534,428 (SstI/SacI); U.S. Pat. No.5,202,248 (NcoI); U.S. Pat. No. 5,139,942 (NdeI); and U.S. Pat. No.5,098,839 (Pad). See also Wilson, G. G., Nucl. Acids Res. 19:2539-2566(1991); and Lunnen, K. D., et al., Gene 74:25-32 (1988).

In the second form, segment D carries a Selectable marker. The toxicgene would eliminate transformants harboring the Vector Donor,Cointegrate, and Byproduct molecules, while the Selectable marker can beused to select for cells containing the Product and against cellsharboring only the Insert Donor.

The third form selects for cells that have both segments A and D in cison the same molecule, but not for cells that have both segments in transon different molecules. This could be embodied by a Selectable markerthat is split into two inactive fragments, one each on segments A and D.

The fragments are so arranged relative to the recombination sites thatwhen the segments are brought together by the recombination event, theyreconstitute a functional Selectable marker. For example, therecombinational event can link a promoter with a structural nucleic acidmolecule (e.g., a gene), can link two fragments of a structural nucleicacid molecule, or can link nucleic acid molecules that encode aheterodimeric gene product needed for survival, or can link portions ofa replicon.

Site-specific recombinase: As used herein, a site specific recombinaseis a type of recombinase which typically has at least the following fouractivities (or combinations thereof): (1) recognition of one or twospecific nucleic acid sequences; (2) cleavage of said sequence orsequences; (3) topoisomerase activity involved in strand exchange; and(4) ligase activity to reseal the cleaved strands of nucleic acid. SeeSauer, B., Current Opinions in Biotechnology 5:521-527 (1994).Conservative site-specific recombination is distinguished fromhomologous recombination and transposition by a high degree ofspecificity for both partners. The strand exchange mechanism involvesthe cleavage and rejoining of specific nucleic acid sequences in theabsence of DNA synthesis (Landy, A. (1989) Ann. Rev. Biochem.58:913-949).

Vector: As used herein, a vector is a nucleic acid molecule (preferablyDNA) that provides a useful biological or biochemical property to anInsert. Examples include plasmids, phages, autonomously replicatingsequences (ARS), centromeres, and other sequences which are able toreplicate or be replicated in vitro or in a host cell, or to convey adesired nucleic acid segment to a desired location within a host cell. AVector can have one or more restriction endonuclease recognition sitesat which the sequences can be cut in a determinable fashion without lossof an essential biological function of the vector, and into which anucleic acid fragment can be spliced in order to bring about itsreplication and cloning. Vectors can further provide primer sites, e.g.,for PCR, transcriptional and/or translational initiation and/orregulation sites, recombinational signals, replicons, Selectablemarkers, etc. Clearly, methods of inserting a desired nucleic acidfragment which do not require the use of recombination, transpositionsor restriction enzymes (such as, but not limited to, UDG cloning of PCRfragments (U.S. Pat. No. 5,334,575, entirely incorporated herein byreference), TA Cloning® brand PCR cloning (Invitrogen Corporation,Carlsbad, Calif.) (also known as direct ligation cloning), and the like)can also be applied to clone a fragment into a cloning vector to be usedaccording to the present invention. The cloning vector can furthercontain one or more selectable markers suitable for use in theidentification of cells transformed with the cloning vector.

Subcloning vector: As used herein, a subcloning vector is a cloningvector comprising a circular or linear nucleic acid molecule whichincludes preferably an appropriate replicon. In the present invention,the subcloning vector (segment D in FIG. 1) can also contain functionaland/or regulatory elements that are desired to be incorporated into thefinal product to act upon or with the cloned nucleic acid Insert(segment A in FIG. 1). The subcloning vector can also contain aSelectable marker (preferably DNA).

Vector Donor: As used herein, a Vector Donor is one of the two parentalnucleic acid molecules (e.g. RNA or DNA) of the present invention whichcarries the nucleic acid segments comprising the nucleic acid vectorwhich is to become part of the desired Product. The Vector Donorcomprises a subcloning vector D (or it can be called the cloning vectorif the Insert Donor does not already contain a cloning vector) and asegment C flanked by recombination sites (see FIG. 1). Segments C and/orD can contain elements that contribute to selection for the desiredProduct daughter molecule, as described above for selection schemes. Therecombination signals can be the same or different, and can be actedupon by the same or different recombinases. In addition, the VectorDonor can be linear or circular.

Primer: As used herein, a primer is a single stranded or double strandedoligonucleotide that is extended by covalent bonding of nucleotidemonomers during amplification or polymerization of a nucleic acidmolecule (e.g. a DNA molecule). In one aspect, the primer may be asequencing primer (for example, a universal sequencing primer). Inanother aspect, the primer may comprise a recombination site or portionthereof

Template: As used herein, a template is a double stranded or singlestranded nucleic acid molecule which is to be amplified, synthesized orsequenced. In the case of a double-stranded DNA molecule, denaturationof its strands to form a first and a second strand is preferablyperformed before these molecules may be amplified, synthesized orsequenced, or the double stranded molecule may be used directly as atemplate. For single stranded templates, a primer complementary to atleast a portion of the template is hybridized under appropriateconditions and one or more polypeptides having polymerase activity (e.g.DNA polymerases and/or reverse transcriptases) may then synthesize amolecule complementary to all or a portion of the template.Alternatively, for double stranded templates, one or moretranscriptional regulatory sequences (e.g., one or more promoters) maybe used in combination with one or more polymerases to make nucleic acidmolecules complementary to all or a portion of the template. The newlysynthesized molecule, according to the invention, may be of equal orshorter length compared to the original template. Mismatch incorporationor strand slippage during the synthesis or extension of the newlysynthesized molecule may result in one or a number of mismatched basepairs. Thus, the synthesized molecule need not be exactly complementaryto the template. Additionally, a population of nucleic acid templatesmay be used during synthesis or amplification to produce a population ofnucleic acid molecules typically representative of the original templatepopulation.

Incorporating: As used herein, incorporating means becoming a part of anucleic acid (e.g., DNA) molecule or primer.

Library: As used herein, a library is a collection of nucleic acidmolecules (circular or linear). In one embodiment, a library maycomprise a plurality (i.e., two or more) of nucleic acid molecules,which may or may not be from a common source organism, organ, tissue, orcell. In another embodiment, a library is representative of all or aportion or a significant portion of the nucleic acid content of anorganism (a “genomic” library), or a set of nucleic acid moleculesrepresentative of all or a portion or a significant portion of theexpressed nucleic acid molecules (a cDNA library or segments derivedtherefrom) in a cell, tissue, organ or organism. A library may alsocomprise random sequences made by de novo synthesis, mutagenesis of oneor more sequences and the like. Such libraries may or may not becontained in one or more vectors.

Amplification: As used herein, amplification is any in vitro method forincreasing a number of copies of a nucleotide sequence with the use ofone or more polypeptides having polymerase activity (e.g., one or morenucleic acid polymerases or one or more reverse transcriptases). Nucleicacid amplification results in the incorporation of nucleotides into aDNA and/or RNA molecule or primer thereby forming a new nucleic acidmolecule complementary to a template. The formed nucleic acid moleculeand its template can be used as templates to synthesize additionalnucleic acid molecules. As used herein, one amplification reaction mayconsist of many rounds of nucleic acid replication. DNA amplificationreactions include, for example, polymerase chain reaction (PCR). One PCRreaction may consist of 5 to 100 cycles of denaturation and synthesis ofa DNA molecule.

Nucleotide: As used herein, a nucleotide is a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid molecule(DNA and RNA). The term nucleotide includes ribonucleoside triphosphatesATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP,dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivativesinclude, for example, [S]dATP, 7-deaza-dGTP and 7-deaza-dATP. The termnucleotide as used herein also refers to dideoxyribonucleosidetriphosphates (ddNTPs) and their derivatives. Illustrated examples ofdideoxyribonucleoside triphosphates include, but are not limited to,ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the presentinvention, a “nucleotide” may be unlabeled or detectably labeled by wellknown techniques. Detectable labels include, for example, radioactiveisotopes, fluorescent labels, chemiluminescent labels, bioluminescentlabels and enzyme labels.

Nucleic acid molecule: As used herein, a nucleic acid molecule is asequence of contiguous nucleotides (riboNTPs, dNTPs or ddNTPs, orcombinations thereof) of any length, which may encode a full-lengthpolypeptide or a fragment of any length thereof, or which may benon-coding. As used herein, the terms “nucleic acid molecule” and“polynucleotide” may be used interchangeably.

Oligonucleotide: As used herein, an oligonucleotide is a synthetic ornatural molecule comprising a covalently linked sequence of nucleotideswhich are joined by a phosphodiester bond between the 3′ position of thepentose of one nucleotide and the 5′ position of the pentose of theadjacent nucleotide.

Polypeptide: As used herein, a polypeptide is a sequence of contiguousamino acids, of any length. As used herein, the terms “peptide,”“oligopeptide,” or “protein” may be used interchangeably with the term“polypeptide.”

Hybridization: As used herein, the terms hybridization and hybridizingrefer to base pairing of two complementary single-stranded nucleic acidmolecules (RNA and/or DNA) to give a double stranded molecule. As usedherein, two nucleic acid molecules may be hybridized, although the basepairing is not completely complementary. Accordingly, mismatched basesdo not prevent hybridization of two nucleic acid molecules provided thatappropriate conditions, well known in the art, are used. In someaspects, hybridization is said to be under “stringent conditions.” By“stringent conditions” as used herein is meant overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

Other terms used in the fields of recombinant nucleic acid technologyand molecular and cell biology as used herein will be generallyunderstood by one of ordinary skill in the applicable arts.

Overview

The present invention relates to methods, compositions and kits for therecombinational and/or topoisomerase-mediated joining of two or moresegments or molecules of nucleic acid or other molecules and/orcompounds (or combinations thereof). The invention also relates toattaching such linked nucleic acids or other molecules and/or compoundsto one or more supports or structures preferably through recombinationsites (which may include recombination protein recognition sequences,topoisomerase recognition sequences, etc.) or portions thereof. Thus,the invention generally relates to linking any number of nucleic acidsor other molecules and/or compounds via nucleic acid linkers comprisingone or more topoisomerase recognition sites and/or one or morerecombination sites or portions thereof. The linked products produced bythe invention may comprise any number of the same or different nucleicacids or other molecules and/or compounds, depending on the startingmaterials. Such starting materials include, but are not limited to, anynucleic acids (or derivatives thereof such as peptide nucleic acids(PNAs)), chemical compounds, detectably labeled molecules (such asfluorescent molecules and chemiluminescent molecules), drugs, peptidesor proteins, lipids, carbohydrates and other molecules and/or compoundscomprising one or more recombination sites or portions thereof. Throughrecombination of such recombination sites and/or topoisomerase-mediatedjoining reactions according to the invention, any number or combinationof such starting molecules and/or compounds can be linked to make linkedproducts of the invention. In addition, deletion or replacement ofcertain portions or components of the linked products of the inventioncan be accomplished by recombination.

In some embodiments, the joined segments may be inserted into adifferent nucleic acid molecule such as vectors, such as byrecombinational cloning methods and/or topoisomerase-mediated joiningmethods of the invention. Thus, in some embodiments, the presentinvention relates to the construction of nucleic acid molecules (RNA orDNA) by combining two or more segments of nucleic acid by arecombination reaction and/or a topoisomerase-mediated joining reactionand inserting the joined two or more segments into a vector byrecombinational cloning. In embodiments where the joined nucleic acidmolecules are to be further combined with an additional nucleic acidmolecule by a recombination reaction, the timing of the tworecombination events, i.e. the joining of the segments and the insertionof the segments into a vector, is not critical. That is to say, it isnot critical to the present invention whether the two or more nucleicacid segments are joined together before insertion into the vector orwhether, for example, one recombination site on each segment firstreacts with a recombination site on the vector and subsequently therecombination sites on the nucleic acid segments react with each otherto join the segments. Moreover, the nucleic acid segments can be clonedin any one or a number of positions within the vector and do not need tobe inserted adjacent to each other, although, in some embodiments,joining of two or more of such segments within the vector is preferred.In accordance with the invention, recombinational cloning allowsefficient selection and identification of molecules (particularlyvectors) containing the combined nucleic acid segments. Thus, two ormore nucleic acid segments of interest can be combined and, optionally,inserted into a single vector suitable for further manipulation of thecombined nucleic acid molecule.

In additional embodiments, at least two (e.g., 2, 3, 4, 5, 6, 7, 8,etc.) nucleic acid segments, each comprising at least one (e.g., 1, 2,3, 4, 5, 6, 7, 8, etc.) recombination site and optionally with at leastone (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) topoisomerase recognition site,are contacted with suitable recombination proteins and/or withtopoisomerase to effect the joining all or a portion of the twomolecules, depending on the position of the recombination sites in themolecules. In certain such embodiments, such as in nucleic acidmolecules comprising at least two recombination sites, at least one ofthe two recombination sites flanks each end of a topoisomeraserecognition site in the molecule. By a recombination site (or atopoisomerase recognition site) that “flanks” another recognition site(e.g., another recombination site or topoisomerase recognition site) ismeant that the two sites are within about 20 nucleotides of each other,or within about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, 1 or 0 nucleotides of each other. Each individual nucleicacid segment may comprise a variety of sequences including, but notlimited to sequences suitable for use as primer sites (e.g., sequencesfor which a primer such as a sequencing primer or amplification primermay hybridize to initiate nucleic acid synthesis, amplification orsequencing), transcription or translation signals or regulatorysequences such as promoters, ribosomal binding sites, Kozak sequences,and start codons, termination signals such as stop codons, origins ofreplication, recombination sites (or portions thereof), topoisomeraserecognition sites (or portions thereof), selectable markers, and genesor portions of genes to create protein fusions (e.g., N-terminal orcarboxy terminal) such as GST, GUS, GFP, 6 histidines, epitopes haptensand the like and combinations thereof. The vectors used for cloning suchsegments may also comprise these functional sequences (e.g., promoters,primer sites etc.). After combination of the segments comprising suchsequences and optimally the cloning of the sequences into one or morevectors, the molecules may be manipulated in a variety of ways includingsequencing or amplification of the target sequence (i.e., by using atleast one or the primer sites introduced by the integration sequence),mutation of the target sequence (i.e., by insertion, deletion orsubstitution in or on the target sequences), and protein expression fromthe target sequence or portions thereof (i.e., by expression oftranslation and/or transcription signals contained by the segmentsand/or vectors).

The present invention also relates to the generation of combinatoriallibraries using the recombinational cloning methods disclosed. Thus, oneor more of the nucleic acid segments joined may comprise a nucleic acidlibrary. Such a library may comprise, for example, nucleic acidsequences corresponding to permutations of a sequence coding for apeptide, polypeptide or protein sequence. The permutations can be joinedto another nucleic acid segment consisting of a single sequence or,alternatively, the second nucleic acid segment may also be a librarycorresponding to permutation of another peptide, polypeptide or proteinsequence such that joining of the two segments may produce a libraryrepresenting all possible combinations of all the permutations of thetwo peptide, polypeptide or proteins sequences. Numerous examples of theuse of combinatorial libraries are known in the art. See, for example,Waterhouse, et al., Nucleic Acids Research, 1993, Vol. 21, No. 9,2265-2266, Tsurushita, et al., Gene, 1996, Vol. 172 No. 1, 59-63,Persson, Int Rev Immunol 1993 10:2-3 153-63, Chanock, et al., InfectAgents Dis 1993 June 2:3 118-31, Burioni, et al., Res Virol 1997March-April 148:2 161-4, Leung, Thromb Haemost 1995 July 74:1 373-6,Sandhu, Crit Rev Biotechnol 1992 12:5-6 437-62 and U.S. Pat. Nos.5,733,743, 5,871,907 and 5,858,657 all of which are specificallyincorporated herein by reference.

Recombination Sites

Recombination sites for use in the invention may be any nucleic acidsequence that can serve as a substrate in a recombination reaction. Suchrecombination sites may be wild-type or naturally occurringrecombination sites or modified or mutant recombination sites. Examplesof recombination sites for use in the invention include, but are notlimited to, phage-lambda recombination sites (such as attP, attB, attL,and attR and mutants or derivatives thereof) and recombination sitesfrom other bacteriophage such as phi80, P22, P2, 186, P4 and P1(including lox sites such as loxp and loxP511). Novel mutated att sites(e.g., attB 1-10, attP 1-10, attR 1-10 and attL 1-10) are described inprevious patent application Ser. No. 60/136,744, filed May 28, 1999,which is specifically incorporated herein by reference. Otherrecombination sites having unique specificity (i.e., a first site willrecombine with its corresponding site and will not recombine with asecond site having a different specificity) are known to those skilledin the art and may be used to practice the present invention. Othersuitable recombination proteins and mutant, modified, variant, orderivative recombination sites for use in the invention include thosedescribed in U.S. Pat. Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969,and 6,277,608 and in U.S. application Ser. No. 09/438,358 (filed Nov.12, 1999), based upon U.S. provisional application No. 60/108,324 (filedNov. 13, 1998). Mutated att sites (e.g., attB 1-10, attP 1-10, attR 1-10and attL 1-10) are described in U.S. provisional patent application No.60/122,389, filed Mar. 2, 1999, 60/126,049, filed Mar. 23, 1999,60/169,983, filed Dec. 10, 1999, and 60/188,000, filed Mar. 9, 2000, andin U.S. application Ser. No. 09/517,466, filed Mar. 2, 2000, and Ser.No. 09/732,914, filed Dec. 11, 2000 (published as 20020007051-A1) thedisclosures of which are specifically incorporated herein by referencein their entirety. Other suitable recombination sites and proteins arethose associated with the GATEWAY™ Cloning Technology available fromInvitrogen Corporation, Carlsbad, Calif., and described in the productliterature of the GATEWAY™ Cloning Technology, the entire disclosures ofall of which are specifically incorporated herein by reference in theirentireties.

Sites that may be used in the present invention include att sites. The15 bp core region of the wildtype att site (GCTTTTTTAT ACTAA (SEQ IDNO:81)), which is identical in all wildtype att sites, may be mutated inone or more positions. The inventors have determined that att sites thatspecifically recombine with other att sites can be constructed byaltering nucleotides in and near the 7 base pair overlap region, bases6-12 of the core region. Thus, recombination sites suitable for use inthe methods, compositions, and vectors of the invention include, but arenot limited to, those with insertions, deletions or substitutions ofone, two, three, four, or more nucleotide bases within the 15 base paircore region (see U.S. application Ser. No. 08/663,002, filed Jun. 7,1996 (now U.S. Pat. No. 5,888,732) and Ser. No. 09/177,387, filed Oct.23, 1998, which describes the core region in further detail, and thedisclosures of which are incorporated herein by reference in theirentireties). Recombination sites suitable for use in the methods,compositions, and vectors of the invention also include those withinsertions, deletions or substitutions of one, two, three, four, or morenucleotide bases within the 15 base pair core region that are at least50% identical, at least 55% identical, at least 60% identical, at least65% identical, at least 70% identical, at least 75% identical, at least80% identical, at least 85% identical, at least 90% identical, or atleast 95% identical to this 15 base pair core region.

Analogously, the core regions in attB1, attP1, attL1 and attR1 areidentical to one another, as are the core regions in attB2, attP2, attL2and attR2. Nucleic acid molecules suitable for use with the inventionalso include those comprising insertions, deletions or substitutions ofone, two, three, four, or more nucleotides within the seven base pairoverlap region (TTTATAC, bases 6-12 in the core region). The overlapregion is defined by the cut sites for the integrase protein and is theregion where strand exchange takes place. Examples of such mutants,fragments, variants and derivatives include, but are not limited to,nucleic acid molecules in which (1) the thymine at position 1 of theseven by overlap region has been deleted or substituted with a guanine,cytosine, or adenine; (2) the thymine at position 2 of the seven byoverlap region has been deleted or substituted with a guanine, cytosine,or adenine; (3) the thymine at position 3 of the seven by overlap regionhas been deleted or substituted with a guanine, cytosine, or adenine;(4) the adenine at position 4 of the seven by overlap region has beendeleted or substituted with a guanine, cytosine, or thymine; (5) thethymine at position 5 of the seven by overlap region has been deleted orsubstituted with a guanine, cytosine, or adenine; (6) the adenine atposition 6 of the seven by overlap region has been deleted orsubstituted with a guanine, cytosine, or thymine; and (7) the cytosineat position 7 of the seven by overlap region has been deleted orsubstituted with a guanine, thymine, or adenine; or any combination ofone or more such deletions and/or substitutions within this seven byoverlap region. The nucleotide sequences of the above described sevenbase pair core regions are set out below in Table 1.

Altered att sites have been constructed that demonstrate that (1)substitutions made within the first three positions of the seven basepair overlap (TTTATAC) strongly affect the specificity of recombination,(2) substitutions made in the last four positions (TTTATAC) onlypartially alter recombination specificity, and (3) nucleotidesubstitutions outside of the seven by overlap, but elsewhere within the15 base pair core region, do not affect specificity of recombination butdo influence the efficiency of recombination. Thus, nucleic acidmolecules and methods of the invention include those comprising oremploying one, two, three, four, five, six, eight, ten, or morerecombination sites which affect recombination specificity, particularlyone or more (e.g., one, two, three, four, five, six, eight, ten, twenty,thirty, forty, fifty, etc.) different recombination sites that maycorrespond substantially to the seven base pair overlap within the 15base pair core region, having one or more mutations that affectrecombination specificity. Particularly preferred such molecules maycomprise a consensus sequence such as NNNATAC wherein “N” refers to anynucleotide (ie., may be A, G, T/U or C). Preferably, if one of the firstthree nucleotides in the consensus sequence is a T/U, then at least oneof the other two of the first three nucleotides is not a T/U.

The core sequence of each att site (attB, attP, attL and attR) can bedivided into functional units consisting of integrase binding sites,integrase cleavage sites and sequences that determine specificity.Specificity determinants are defined by the first three positionsfollowing the integrase top strand cleavage site. These three positionsare shown with underlining in the following reference sequence:CAACTTTTTTATAC AAAGTTG (SEQ ID NO:82). Modification of these threepositions (64 possible combinations) can be used to generate att sitesthat recombine with high specificity with other att sites having thesame sequence for the first three nucleotides of the seven base pairoverlap region. The possible combinations of first three nucleotides ofthe overlap region are shown in Table 1.

TABLE 1 Modifications of the First Three Nucleotides of the att SiteSeven Base Pair Overlap Region that Alter Recombination Specificity. AAACAA GAA TAA AAC CAC GAC TAC CAG GAG TAG AAG CAT GAT TAT AAT CCA GCA TCAACA CCC GCC TCC ACC CCG GCG TCG ACG CCT GCT TCT ACT CGA GGA TGA AGA CGCGGC TGC AGC CGG GGG TGG AGG CGT GGT TGT AGT CTA GTA TTA ATA CTC GTC TTCATC CTG GTG TTG ATG CTT GTT TTT ATT

Representative examples of seven base pair att site overlap regionssuitable for in methods, compositions and vectors of the invention areshown in Table 2. The invention further includes nucleic acid moleculescomprising one or more (e.g., one, two, three, four, five, six, eight,ten, twenty, thirty, forty, fifty, etc.) nucleotides sequences set outin Table 2. Thus, for example, in one aspect, the invention providesnucleic acid molecules comprising the nucleotide sequence GAAATAC,GATATAC, ACAATAC, or TGCATAC.

TABLE 2 Representative Examples of Seven Base Pair att Site OverlapRegions Suitable for use in the recombination sites of the Invention.AAAATAC CAAATAC GAAATAC TAAATAC AACATAC CACATAC GACATAC TACATAC AAGATACCAGATAC GAGATAC TAGATAC AATATAC CATATAC GATATAC TATATAC ACAATAC CCAATACGCAATAC TCAATAC ACCATAC CCCATAC GCCATAC TCCATAC ACGATAC CCGATAC GCGATACTCGATAC ACTATAC CCTATAC GOTATAC TCTATAC AGAATAC CGAATAC GGAATAC TGAATACAGCATAC CGCATAC GGCATAC TGCATAC AGGATAC CGGATAC GGGATAC TGGATAC AGTATACCGTATAC GGTATAC TGTATAC ATAATAC CTAATAC GTAATAC TTAATAC ATCATAC CTCATACGTCATAC TTCATAC ATGATAC CTGATAC GTGATAC TTGATAC ATTATAC CTTATAC GTTATACTTTATAC

As noted above, alterations of nucleotides located 3′ to the three basepair region discussed above can also affect recombination specificity.For example, alterations within the last four positions of the sevenbase pair overlap can also affect recombination specificity.

For example, mutated att sites that may be used in the practice of thepresent invention include attB1 (AGCCTGCTTT TTTGTACAAA CTTGT (SEQ IDNO:83)), attP1 (TACAGGTCAC TAATACCATC TAAGTAGTTG ATTCATAGTG ACTGGATATGTTGTGTTTTA CAGTATTATG TAGTCTGTTT TTTATGCAAA ATCTAATTTA ATATATTGATATTTATATCA TTTTACGTTT CTCGTTCAGC TTTTTTGTAC AAAGTTGGCA TTATAAAAAAGCATTGCTCA TCAATTTGTT GCAACGAACA GGTCACTATC AGTCAAAATA AAATCATTAT TTG(SEQ ID NO:84)), attL1 (CAAATAATGA TTTTATTTTG ACTGATAGTG ACCTGTTCGTTGCAACAAAT TGATAAGCAA TGCTTTTTTA TAATGCCAAC TTTGTACAAA AAAGCAGGCT (SEQID NO:85)), and attR1 (ACAAGTTTGT ACAAAAAAGC TGAACGAGAA ACGTAAAATGATATAAATAT CAATATATTA AATTAGATTT TGCATAAAAA ACAGACTACA TAATACTGTAAAACACAACA TATCCAGTCA CTATG (SEQ ID NO:86)). Table 3 provides thesequences of the regions surrounding the core region for the wild typeatt sites (attB0, P0, R0, and L0) as well as a variety of other suitablerecombination sites. Those skilled in the art will appreciated that theremainder of the site is the same as the corresponding site (B, P, L, orR) listed above.

TABLE 3 Nucleotide sequences of representative att sites. attB0AGCCTGCTTT TTTATACTAA CTTG (SEQ ID NO: 87) AGC attP0 GTTCAGCTTTTTTATACTAA GTTG (SEQ ID NO: 88) GCA attL0 AGCCTGCTTT TTTATACTAA GTTG(SEQ ID NO: 89) GCA attR0 GTTCAGCTTT TTTATACTAA CTTG (SEQ ID NO: 90) AGCattB1 AGCCTGCTTT TTTGTACAAA (SEQ ID NO: 83) CTTGT attP1 GTTCAGCTTTTTTGTACAAA (SEQ ID NO: 91) GTTGGCA attL1 AGCCTGCTTT TTTGTACAAA (SEQ IDNO: 92) GTTGGCA attR1 GTTCAGCTTT TTTGTACAAA (SEQ ID NO: 93) CTTGT attB2ACCCAGCTTT CTTGTACAAA (SEQ ID NO: 94) attP2 GTTCAGCTTT CTTGTACAAA (SEQID NO: 95) GTTGGCA attL2 ACCCAGCTTT CTTGTACAAA (SEQ ID NO: 96) GTTGGCAattR2 GTTCAGCTTT CTTGTACAAA (SEQ ID NO: 97) GTGGT attB5 CAACTTTATTATACAAAGTT GT (SEQ ID NO: 98) attP5 GTTCAACTTT ATTATACAAA (SEQ ID NO:99) GTTGGCA attL5 CAACTTTATT ATACAAAGTT GGCA (SEQ ID NO: 100) attR5GTTCAACTTT ATTATACAAA (SEQ ID NO: 101) GTTGT attB11 CAACTTTTCTATACAAAGTT GT (SEQ ID NO: 102) attP11 GTTCAACTTT TCTATACAAA (SEQ ID NO:103) GTTGGCA attL11 CAACTTTTCT ATACAAAGTT GGCA (SEQ ID NO: 104) attR11GTTCAACTTT TCTATACAAA (SEQ ID NO: 105) GTTGT attB17 CAACTTTTGTATACAAAGTT GT (SEQ ID NO: 106) attP17 GTTCAACTTT TGTATACAAA (SEQ ID NO:107) GTTGGCA attL17 CAACTTTTGT ATACAAAGTT GGCA (SEQ ID NO: 108) attR17GTTCAACTTT TGTATACAAA GTTGT (SEQ ID NO: 109) attB19 CAACTTTTTCGTACAAAGTT GT (SEQ ID NO: 110) attP19 GTTCAACTTT TTCGTACAAA (SEQ ID NO:111) GTTGGCA attL19 CAACTTTTTC GTACAAAGTT GGCA (SEQ ID NO: 112) attR19GTTCAACTTT TTCGTACAAA GTTGT (SEQ ID NO: 113) attB20 CAACTTTTTGGTACAAAGTT GT (SEQ ID NO: 114) attP20 GTTCAACTTT TTGGTACAAA (SEQ ID NO:115) GTTGGCA attL20 CAACTTTTTG GTACAAAGTT GGCA (SEQ ID NO: 116) attR20GTTCAACTTT TTGGTACAAA GTTGT (SEQ ID NO: 117) attB21 CAACTTTTTAATACAAAGTT GT (SEQ ID NO: 118) attP21 GTTCAACTTT TTAATACAAA (SEQ ID NO:119) GTTGGCA attL21 CAACTTTTTA ATACAAAGTT GGCA (SEQ ID NO: 120) attR21GTTCAACTTT TTAATACAAA GTTGT (SEQ ID NO: 121)

Other recombination sites having unique specificity (i.e., a first sitewill recombine with its corresponding site and will not recombine with asecond site having a different specificity) are known to those skilledin the art and may be used to practice the present invention.Corresponding recombination proteins for these systems may be used inaccordance with the invention with the indicated recombination sites.Other systems providing recombination sites and recombination proteinsfor use in the invention include the FLP/FRT system from Saccharomycescerevisiae, the resolvase family (e.g., γδ, TndX, TnpX, Tn3 resolvase,Hin, Hjc, Gin, SpCCE1, ParA, and Cin), and IS231 and other Bacillusthuringiensis transposable elements. Other suitable recombinationsystems for use in the present invention include the XerC and XerDrecombinases and the psi, dif and cer recombination sites in E. coli.Other suitable recombination sites may be found in U.S. Pat. No.5,851,808 issued to Elledge and Liu which is specifically incorporatedherein by reference.

Recombination sites used with the invention may also have embeddedfunctions or properties. An embedded functionality is a function orproperty conferred by a nucleotide sequence in a recombination site thatis not directly associated with recombination efficiency or specificity.For example, recombination sites may contain protein coding sequences(e.g. intein coding sequences), intron/exon splice sites, origins ofreplication, and/or stop codons. Further, recombination sites that havemore than one (e.g., two, three, four, five, etc.) embedded functions orproperties may also be prepared.

In some instances it will be advantageous to remove either RNAcorresponding to recombination sites from RNA transcripts or amino acidresidues encoded by recombination sites from polypeptides translatedfrom such RNAs. Removal of such sequences can be performed in severalways and can occur at either the RNA or protein level. One instancewhere it may be advantageous to remove RNA transcribed from arecombination site will be when constructing a fusion polypeptidebetween a polypeptide of interest and a coding sequence present on thevector. The presence of an intervening recombination site between theORF of the polypeptide of interest and the vector coding sequences mayresult in the recombination site (1) contributing codons to the mRNAthat result in the inclusion of additional amino acid residues in theexpression product, (2) contributing a stop codon to the mRNA thatprevents the production of the desired fusion protein, and/or (3)shifting the reading frame of the mRNA such that the two protein are notfused “in-frame.”

In one aspect, the invention provides methods for removing nucleotidesequences encoded by recombination sites from RNA molecules. One exampleof such a method employs the use of intron/exon splice sites to removeRNA encoded by recombination sites from RNA transcripts. Nucleotidesequences that encode intron/exon splice sites may be fully or partiallyembedded in the recombination sites used in the present invention and/ormay encoded by adjacent nucleic acid sequence. Sequences to be excisedfrom RNA molecules may be flanked by splice sites that are appropriatelylocated in the sequence of interest and/or on the vector. For example,one intron/exon splice site may be encoded by a recombination site andanother intron/exon splice site may be encoded by other nucleotidesequences (e.g., nucleic acid sequences of the vector or a nucleic acidof interest). Nucleic acid splicing is well known to those skilled inthe art and is discussed in the following publications: R. Reed, Curr.Opin. Genet. Devel. 6:215-220 (1996); S. Mount, Nucl. Acids. Res.10:459-472, (1982); P. Sharp, Cell 77:805-815, (1994); K. Nelson and M.Green, Genes and Devel. 23:319-329 (1988); and T. Cooper and W. Mattox,Am. J. Hum. Genet. 61:259-266 (1997).

Splice sites can be suitably positioned in a number of locations. Forexample, a Destination Vector designed to express an inserted ORF withan N-terminal fusion—for example, with a detectable marker—the firstsplice site could be encoded by vector sequences located 3′ to thedetectable marker coding sequences and the second splice site could bepartially embedded in the recombination site that separates thedetectable marker coding sequences from the coding sequences of the ORF.Further, the second splice site either could abut the 3′ end of therecombination site or could be positioned a short distance (e.g., 2, 4,8, 10, 20 nucleotides) 3′ to the recombination site. In addition,depending on the length of the recombination site, the second splicesite could be fully embedded in the recombination site.

A modification of the method described above involves the connection ofmultiple nucleic acid segments that, upon expression, results in theproduction of a fusion protein. In one specific example, one nucleicacid segment encodes detectable marker—for example, GFP—and anothernucleic acid segment that encodes an ORF of interest. Each of thesesegments is flanked by recombination sites. In addition, the nucleicacid segments that encodes the detectable marker contains an intron/exonsplice site near its 3′ terminus and the nucleic acid segments thatcontains the ORF of interest also contains an intron/exon splice sitenear its 5′ terminus. Upon recombination, the nucleic acid segment thatencodes the detectable marker is positioned 5′ to the nucleic acidsegment that encodes the ORF of interest. Further, these two nucleicacid segments are separated by a recombination site that is flanked byintron/exon splice sites. Excision of the intervening recombination sitethus occurs after transcription of the fusion mRNA. Thus, in one aspect,the invention is directed to methods for removing RNA transcribed fromrecombination sites from transcripts generated from nucleic acidsdescribed herein.

Splice sites may introduced into nucleic acid molecules to be used inthe present invention in a variety of ways. One method that could beused to introduce intron/exon splice sites into nucleic acid segments isby the use of PCR. For example, primers could be used to generatenucleic acid segments corresponding to an ORF of interest and containingboth a recombination site and an intron/exon splice site.

The above methods can also be used to remove RNA corresponding torecombination sites when the nucleic acid segment that is recombinedwith another nucleic acid segment encodes RNA that is not produced in atranslatable format. One example of such an instance is where a nucleicacid segment is inserted into a vector in a manner that results in theproduction of antisense RNA. As discussed below, this antisense RNA maybe fused, for example, with RNA that encodes a ribozyme. Thus, theinvention also provides methods for removing RNA corresponding torecombination sites from such molecules.

The invention further provides methods for removing amino acid sequencesencoded by recombination sites from protein expression products byprotein splicing. Nucleotide sequences that encode protein splice sitesmay be fully or partially embedded in the recombination sites thatencode amino acid sequences excised from proteins or protein splicesites may be encoded by adjacent nucleotide sequences. Similarly, oneprotein splice site may be encoded by a recombination site and anotherprotein splice sites may be encoded by other nucleotide sequences (e.g.,nucleic acid sequences of the vector or a nucleic acid of interest).

It has been shown that protein splicing can occur by excision of anintein from a protein molecule and ligation of flanking segments (see,e.g., Derbyshire et al., Proc. Natl. Acad. Sci. (USA) 95:1356-1357(1998)). In brief, inteins are amino acid segments that arepost-translationally excised from proteins by a self-catalytic splicingprocess. A considerable number of intein consensus sequences have beenidentified (see, e.g., Perler, Nucleic Acids Res. 27:346-347 (1999)).

Similar to intron/exon splicing, N- and C-terminal intein motifs havebeen shown to be involved in protein splicing. Thus, the inventionfurther provides compositions and methods for removing amino acidresidues encoded by recombination sites from protein expression productsby protein splicing. In particular, this aspect of the invention isrelated to the positioning of nucleic acid sequences that encode inteinsplice sites on both the 5′ and 3′ end of recombination sites positionedbetween two coding regions. Thus, when the protein expression product isincubated under suitable conditions, amino acid residues encoded theserecombination sites will be excised.

Protein splicing may be used to remove all or part of the amino acidsequences encoded by recombination sites. Nucleic acid sequence thatencode inteins may be fully or partially embedded in recombination sitesor may adjacent to such sites. In certain circumstances, it may bedesirable to remove considerable numbers of amino acid residues beyondthe N- and/or C-terminal ends of amino acid sequences encoded byrecombination sites. In such instances, intein coding sequence may belocated a distance (e.g., 30, 50, 75, 100, etc. nucleotides) 5′ and/or3′ to the recombination site.

While conditions suitable for intein excision will vary with theparticular intein, as well as the protein that contains this intein,Chong et al., Gene 192:271-281 (1997), have demonstrated that a modifiedSaccharomyces cerevisiae intein, referred to as Sce VMA intein, can beinduced to undergo self-cleavage by a number of agents including1,4-dithiothreitol (DTT), β-mercaptoethanol, and cysteine. For example,intein excision/splicing can be induced by incubation in the presence of30 mM DTT, at 4° C. for 16 hours.

Corresponding recombination proteins for these systems may be used inaccordance with the invention with the indicated recombination sites.Other systems providing recombination sites and recombination proteinsfor use in the invention include the FLP/FRT system from Saccharomycescerevisiae, the resolvase family (e.g., 4, Tn3 resolvase, Hin, Gin andCin), and IS231 and other Bacillus thuringiensis transposable elements.Other suitable recombination systems for use in the present inventioninclude the XerC and XerD recombinases and the psi, dif and cerrecombination sites in E. coli. Other suitable recombination sites maybe found in U.S. Pat. No. 5,851,808 issued to Elledge and Liu which isspecifically incorporated herein by reference. Preferred recombinationproteins and mutant or modified recombination sites for use in theinvention include those described in U.S. Pat. Nos. 5,888,732,6,171,861, 6,143,557, 6,270,969 and 6,277,608, and commonly owned,co-pending U.S. application Ser. No. 09/438,358 (filed Nov. 12, 1999),Ser. No. 09/517,466 (filed Mar. 2, 2000), Ser. No. 09/695,065 (filedOct. 25, 2000) and Ser. No. 09/732,914 (filed Dec. 11, 2000), thedisclosures of all of which are incorporated herein by reference intheir entireties, as well as those associated with the GATEWAY™ CloningTechnology available from Invitrogen Corporation (Carlsbad, Calif.).

Topoisomerase Cloning

The present invention also relates to methods of using one or moretopoisomerases to generate a recombinant nucleic acid molecule from twoor more nucleotide sequences. In a first aspect, the invention providesa method for generating a ds recombinant nucleic acid molecule that iscovalently linked in one strand. Such a method is directed to linking afirst and at least a second nucleotide sequence with at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) topoisomerase (e.g., a type IA,type IB, and/or type II topoisomerase) such that one strand, but notboth strands, is covalently linked (see, for example, FIGS. 11A-F). In asecond aspect, the invention provides a method for generating a dsrecombinant nucleic acid molecule covalently linked in both strands.Such a method is directed to linking a first and at least a secondnucleotide sequence with at least one topoisomerase, such that ligatedends are covalently linked in both strands (i.e., the ds recombinantnucleic acid molecule contain no nicks at the positions where ends wereligated; see, for example, FIGS. 12A-D). In a third aspect, theinvention provides a method for generating a recombinant nucleic acidmolecule covalently linked in one strand, wherein the substratenucleotide sequences linked according to the method include at least onesingle stranded nucleotide sequence, which can be covalently linked to asecond (or more) single stranded nucleotide sequence or to a nucleicacid molecule (see, for example, FIG. 15).

A method for generating a ds recombinant nucleic acid moleculecovalently linked in one strand can be performed by contacting a firstnucleic acid molecule which has a site-specific topoisomeraserecognition site (e.g., a type IA or a type II topoisomerase recognitionsite), or a cleavage product thereof, at a 5′ or 3′ terminus, with asecond (or other) nucleic acid molecule, and optionally, a topoisomerase(e.g., a type IA, type IB, and/or type II topoisomerase), such that thesecond nucleotide sequence can be covalently attached to the firstnucleotide sequence. As disclosed herein, the methods of the inventioncan be performed using any number of nucleotide sequences, typicallynucleic acid molecules wherein at least one of the nucleotide sequenceshas a site-specific topoisomerase recognition site (e.g., a type IA, ortype II topoisomerase), or cleavage product thereof, at one or both 5′termini (see, for example, FIGS. 11A-11F).

A method for generating a ds recombinant nucleic acid moleculecovalently linked in both strands can be performed, for example, bycontacting a first nucleic acid molecule having a first end and a secondend, wherein, at the first end or second end or both, the first nucleicacid molecule has a topoisomerase recognition site (or cleavage productthereof) at or near the 3′ terminus; at least a second nucleic acidmolecule having a first end and a second end, wherein, at the first endor second end or both, the at least second double stranded nucleotidesequence has a topoisomerase recognition site (or cleavage productthereof) at or near a 3′ terminus; and at least one site specifictopoisomerase (e.g., a type IA and/or a type IB topoisomerase), underconditions such that all components are in contact and the topoisomerasecan effect its activity. A covalently linked ds recombinant nucleic acidgenerated according to a method of this aspect of the invention ischaracterized, in part, in that it does not contain a nick in eitherstrand at the position where the nucleic acid molecules are joined. Inone embodiment, the method is performed by contacting a first nucleicacid molecule and a second (or other) nucleic acid molecule, each ofwhich has a topoisomerase recognition site, or a cleavage productthereof, at the 3′ termini or at the 5′ termini of two ends to becovalently linked. In another embodiment, the method is performed bycontacting a first nucleic acid molecule having a topoisomeraserecognition site, or cleavage product thereof, at the 5′ terminus andthe 3′ terminus of at least one end, and a second (or other) nucleicacid molecule having a 3′ hydroxyl group and a 5′ hydroxyl group at theend to be linked to the end of the first nucleic acid moleculecontaining the recognition sites. As disclosed herein, the methods canbe performed using any number of nucleic acid molecules having variouscombinations of termini and ends (see, for example, FIG. 12A-12D).

Topoisomerases are categorized as type I, including type IA and type IBtopoisomerases, which cleave a single strand of a double strandednucleic acid molecule, and type II topoisomerases (gyrases), whichcleave both strands of a nucleic acid molecule. Type IA and IBtopoisomerases cleave one strand of a nucleic acid molecule. Cleavage ofa nucleic acid molecule by type IA topoisomerases generates a 5′phosphate and a 3′ hydroxyl at the cleavage site, with the type IAtopoisomerase covalently binding to the 5′ terminus of a cleaved strand.In comparison, cleavage of a nucleic acid molecule by type IBtopoisomerases generates a 3′ phosphate and a 5′ hydroxyl at thecleavage site, with the type IB topoisomerase covalently binding to the3′ terminus of a cleaved strand. As disclosed herein, type I and type IItopoisomerases, as well as catalytic domains and mutant forms thereof,are useful for generating ds recombinant nucleic acid moleculescovalently linked in both strands according to a method of theinvention.

Type IA topoisomerases include E. coli topoisomerase I, E. colitopoisomerase III, eukaryotic topoisomerase II, archeal reverse gyrase,yeast topoisomerase III, Drosophila topoisomerase III, humantopoisomerase m, Streptococcus pneumoniae topoisomerase III, and thelike, including other type IA topoisomerases (see Berger, Biochim.Biophys. Acta 1400:3-18, 1998; DiGate and Marians, J. Biol. Chem.264:17924-17930, 1989; Kim and Wang, J. Biol. Chem. 267:17178-17185,1992; Wilson et al., J. Biol. Chem. 275:1533-1540, 2000; Hanai et al.,Proc. Natl. Acad. Sci. USA 93:3653-3657, 1996, U.S. Pat. No. 6,277,620,each of which is incorporated herein by reference). E. colitopoisomerase III, which is a type IA topoisomerase that recognizes,binds to and cleaves the sequence 5′-GCAACTT-3′, can be particularlyuseful in a method of the invention (Zhang et al., J. Biol. Chem.270:23700-23705, 1995, which is incorporated herein by reference). Ahomolog, the traE protein of plasmid RP4, has been described by Li etal., J. Biol. Chem. 272:19582-19587 (1997) and can also be used in thepractice of the invention. A DNA-protein adduct is formed with theenzyme covalently binding to the 5′-thymidine residue, with cleavageoccurring between the two thymidine residues.

Type IB topoisomerases include the nuclear type I topoisomerases presentin all eukaryotic cells and those encoded by vaccinia and other cellularpoxviruses (see Cheng et al., Cell 92:841-850, 1998, which isincorporated herein by reference). The eukaryotic type IB topoisomerasesare exemplified by those expressed in yeast, Drosophila and mammaliancells, including human cells (see Caron and Wang, Adv. Pharmacol.29B:271-297, 1994; Gupta et al., Biochim. Biophys. Acta 1262:1-14, 1995,each of which is incorporated herein by reference; see, also, Berger,supra, 1998). Viral type IB topoisomerases are exemplified by thoseproduced by the vertebrate poxviruses (vaccinia, Shope fibroma virus,ORF virus, fowlpox virus, and molluscum contagiosum virus), and theinsect poxvirus (Amsacta moorei entomopoxvirus) (see Shuman, Biochim.Biophys. Acta 1400:321-337, 1998; Petersen et al., Virology 230:197-206,1997; Shuman and Prescott, Proc. Natl. Acad. Sci. USA 84:7478-7482,1987; Shuman, J. Biol. Chem. 269:32678-32684, 1994; U.S. Pat. No.5,766,891; PCT/US95/16099; PCT/US98/12372, each of which is incorporatedherein by reference; see, also, Cheng et al., supra, 1998).

Type II topoisomerases include, for example, bacterial gyrase, bacterialDNA topoisomerase IV, eukaryotic DNA topoisomerase II, and T-even phageencoded DNA topoisomerases (Roca and Wang, Cell 71:833-840, 1992; Wang,J. Biol. Chem. 266:6659-6662, 1991, each of which is incorporated hereinby reference; Berger, supra, 1998). Like the type IB topoisomerases, thetype II topoisomerases have both cleaving and ligating activities. Inaddition, like type IB topoisomerase, substrate nucleic acid moleculescan be prepared such that the type II topoisomerase can form a covalentlinkage to one strand at a cleavage site. For example, calf thymus typeII topoisomerase can cleave a substrate nucleic acid molecule containinga 5′ recessed topoisomerase recognition site positioned threenucleotides from the 5′ end, resulting in dissociation of the threenucleotide sequence 5′ to the cleavage site and covalent binding the ofthe topoisomerase to the 5′ terminus of the nucleic acid molecule(Andersen et al., supra, 1991). Furthermore, upon contacting such a typeII topoisomerase charged nucleic acid molecule with a second nucleotidesequence containing a 3′ hydroxyl group, the type II topoisomerase canligate the sequences together, and then is released from the recombinantnucleic acid molecule. As such, type II topoisomerases also are usefulfor performing methods of the invention.

Structural analysis of topoisomerases indicates that the members of eachparticular topoisomerase families, including type IA, type IB and typeII topoisomerases, share common structural features with other membersof the family (Berger, supra, 1998). In addition, sequence analysis ofvarious type IB topoisomerases indicates that the structures are highlyconserved, particularly in the catalytic domain (Shuman, supra, 1998;Cheng et al., supra, 1998; Petersen et al., supra, 1997). For example, adomain comprising amino acids 81 to 314 of the 314 amino acid vacciniatopoisomerase shares substantial homology with other type IBtopoisomerases, and the isolated domain has essentially the sameactivity as the full length topoisomerase, although the isolated domainhas a slower turnover rate and lower binding affinity to the recognitionsite (see Shuman, supra, 1998; Cheng et. al., supra, 1998). In addition,a mutant vaccinia topoisomerase, which is mutated in the amino terminaldomain (at amino acid residues 70 and 72) displays identical propertiesas the full length topoisomerase (Cheng et al., supra, 1998). In fact,mutation analysis of vaccinia type IB topoisomerase reveals a largenumber of amino acid residues that can be mutated without affecting theactivity of the topoisomerase, and has identified several amino acidsthat are required for activity (Shuman, supra, 1998). In view of thehigh homology shared among the vaccinia topoisomerase catalytic domainand the other type IB topoisomerases, and the detailed mutation analysisof vaccinia topoisomerase, it will be recognized that isolated catalyticdomains of the type IB topoisomerases and type IB topoisomerases havingvarious amino acid mutations can be used in the methods of theinvention.

The various topoisomerases exhibit a range of sequence specificity. Forexample, type II topoisomerases can bind to a variety of sequences, butcleave at a highly specific recognition site (see Andersen et al., J.Biol. Chem. 266:9203-9210, 1991, which is incorporated herein byreference). In comparison, the type IB topoisomerases include sitespecific topoisomerases, which bind to and cleave a specific nucleotidesequence (“topoisomerase recognition site”). Upon cleavage of a nucleicacid molecule by a topoisomerase, for example, a type IB topoisomerase,the energy of the phosphodiester bond is conserved via the formation ofa phosphotyrosyl linkage between a specific tyrosine residue in thetopoisomerase and the 3′ nucleotide of the topoisomerase recognitionsite. Where the topoisomerase cleavage site is near the 3′ terminus ofthe nucleic acid molecule, the downstream sequence (3′ to the cleavagesite) can dissociate, leaving a nucleic acid molecule having thetopoisomerase covalently bound to the newly generated 3′ end (see FIG.29).

A method of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand, can be performed bycontacting 1) a first nucleic acid molecule having a first end and asecond end, wherein the first nucleic acid molecule has a site-specifictopoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) at or near the 5′ terminus of the firstend or the second end or both and, optionally, comprising one or morerecombination sites; 2) at least a second nucleic acid molecule thathas, or can be made to have, a first end and a second end; and 3) atleast one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) site-specifictopoisomerase (e.g., a type IA or a type IB topoisomerase), underconditions such that all components are in contact and the at least onetopoisomerase can effect its activity. For example, the topoisomerasecan be a type IA topoisomerase such as E. coli topoisomerase I, E. colitopoisomerase III, or a eukaryotic topoisomerase III. Upon cleavage of anucleic acid molecule, the topoisomerase preferably is stably bound tothe 5′ terminus. Upon cleavage by the topoisomerase, the cleaved nucleicacid molecule often may comprise a 3′ overhanging sequence. Once nucleicacid molecules are joined by the methods described above, the resultingmolecules may then be used in recombination reactions, such as thosedescribed elsewhere herein.

A method of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand can be performed such that anycombination of ends are linked, and wherein one strand at the ends beinglinked is covalently linked and the other strand is not covalentlylinked, but contains a nick. For example, the first nucleic acidmolecule can comprise a coding sequence, wherein the ATG start codon isat or near the first end and a poly A signal is encoded at or near thesecond end; and a second nucleic acid molecule can comprise a promoterelement, which functions when positioned upstream of a coding sequence,and the first end is upstream of the second end, the method can beperformed wherein a site-specific topoisomerase recognition site (e.g.,a type IA or a type II topoisomerase recognition site) is at or near the5′ terminus of the first end of the first nucleic acid molecule, andwherein the contacting is performed under conditions such that thetopoisomerase (e.g., a type IA or a type II topoisomerase) cancovalently link the 5′ terminus of the first end of the first nucleicacid molecule to the 3′ terminus of the first end of the second nucleicacid molecule, thereby generating a ds recombinant nucleic acidmolecule, in which a polypeptide can be expressed from the codingsequence. Alternatively, the method can be performed wherein thetopoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) is at or near the 5′ terminus of thesecond end of the first nucleic acid molecule, and wherein thecontacting is performed under conditions such that the topoisomerase(e.g., a type IA or a type II topoisomerase recognition site) cancovalently link the 5′ terminus of the second end of the first nucleicacid molecule to the 3′ terminus of the first end of the second nucleicacid molecule, thereby generating a ds recombinant nucleic acid moleculefrom which an antisense molecule can be expressed. Once nucleic acidmolecules are joined by the methods described above, the resultingmolecules may then be used in recombination reactions, such as thosedescribed elsewhere herein.

As another example using the first nucleic acid molecule and secondnucleic acid molecule described above, the method can be performed,wherein the topoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) is at or near the 5′ terminus of each ofthe first end and the second end of the first nucleic acid molecule, andwherein the contacting is performed under conditions such that the typeIA topoisomerase can covalently link the 5′ terminus of the first end ofthe first nucleic acid molecule to the 3′ terminus of the first end ofthe second nucleic acid molecule, and the 5′ terminus of the second endof the first nucleic acid molecule to the 3′ terminus of the second endof the second nucleic acid molecule. As such, the ds recombinant nucleicacid molecule generated by the method is circularized, and includes anick in each strand opposite the location where a strand was covalentlylinked by a topoisomerase (e.g., a type IA or a type II topoisomerase).Furthermore, the promoter of the second nucleic acid molecule caninitiate expression of the first nucleic acid molecule. In oneembodiment, the circularized ds recombinant nucleic acid moleculecomprises a vector. Once nucleic acid molecules are joined by themethods described above, the resulting molecules may then be used incombination reactions, such as those described elsewhere herein.

As another example using the first nucleic acid molecule and secondnucleic acid molecule described above, the method can be performed,wherein the topoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) is at or near the 5′ terminus of each ofthe first end and the second end of the first nucleic acid molecule, andwherein the contacting is performed under conditions such that thetopoisomerase (e.g., a type IA or a type II topoisomerase) cancovalently link the 5′ terminus of the first end of the first nucleicacid molecule to the 3′ terminus of the second end of the second nucleicacid molecule, and the 5′ terminus of the second end of the firstnucleic acid molecule to the 3′ terminus of the first end of the secondnucleic acid molecule. As such, the ds recombinant nucleic acid moleculegenerated by the method is circularized, and includes a nick in eachstrand opposite the location where a strand was covalently linked bytopoisomerase (e.g., a type IA or a type II topoisomerase recognitionsite). Furthermore, the promoter of the second nucleic acid molecule caninitiate expression of an antisense sequence. In one embodiment, thecircularized ds recombinant nucleic acid molecule comprises a vector.Once nucleic acid molecules are joined by the methods described above,the resulting molecules may then be used in recombination reactions,such as those described elsewhere herein.

As disclosed herein, a method of generating a ds recombinant nucleicacid molecule covalently linked in one strand, involving a first nucleicacid molecule and at least a second nucleic acid molecule, can furtherinclude a step for amplifying the ds recombinant nucleic acid moleculecovalently linked in one strand. The amplification reaction can becarried out by contacting the ds recombinant nucleic acid molecule withan amplification reaction primer pair, wherein a first primer of thepair is capable of binding to the covalently linked strand, at or nearone end of the first or second nucleic acid molecule, and priming anamplification reaction toward the other nucleic acid molecule togenerate a first extension product that is identical in nucleotidesequence to the nicked strand of the ds recombinant nucleic acidmolecule; and the second primer of the pair is capable of binding to thefirst extension product, typically at or near the 3′ terminus, and, inthe presence of the first primer, can generate an amplification productusing the covalently linked strand and the extension product (orextension products generated therefrom) as templates. For example, themethod can be performed such that the type IA topoisomerase recognitionsite is at or near a first end of the first nucleic acid molecule, andthe method further includes contacting the ds recombinant nucleic acidmolecule with an amplification reaction primer pair, wherein a forwardprimer is capable of binding at or near the second end of the firstnucleic acid molecule, and wherein a reverse primer is capable ofbinding to a nucleotide sequence complementary to at least a portion ofthe second end of the second nucleic acid molecule; and amplifying theds recombinant nucleic acid molecule. The first nucleic acid moleculecan include a coding region and the second nucleic acid molecule caninclude a regulatory element. Once nucleic acid molecules are joined bythe methods described above, the resulting molecules may then be used inrecombination reactions, such as those described elsewhere herein.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in one strand also can be performed by contacting 1) a firstnucleic acid molecule having a first end and a second end, wherein thefirst nucleic acid molecule has a site-specific topoisomeraserecognition site (e.g., a type IA or a type II topoisomerase recognitionsite) at or near the 5′ terminus of the first end or the second end orboth; 2) at least a second nucleic acid molecule that has, or can bemade to have, a first end and a second end; 3) at least a third nucleicacid molecule which has, or can be made to have, a first end and asecond end, each end further comprising a 5′ terminus and a 3′ terminus;and 4) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)site-specific topoisomerase (e.g., a type IA or a type IItopoisomerase), under conditions such that all components are in contactand the at least one topoisomerase can effect its activity. For example,the topoisomerase can be a type IA topoisomerase such as E. colitopoisomerase I, E. coli topoisomerase III, or a eukaryotictopoisomerase III. Upon cleavage of a nucleic acid molecule, thetopoisomerase preferably is stably bound to the 5′ terminus. Preferably,upon cleavage by the topoisomerase, the cleaved nucleic acid moleculecomprises a 3′ overhanging sequence. Once nucleic acid molecules arejoined by the methods described above, the resulting molecules may thenbe used in recombination reactions, such as those described elsewhereherein.

A method of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one strand, involving a first nucleic acidmolecule that contains a site-specific topoisomerase recognition site(e.g., a type IA or a type IB topoisomerase recognition site), orcleavage product thereof, at least a second nucleic acid molecule, andat least a third nucleic acid molecule can be performed such that anycombination of ends are linked, and one strand at the ends being linkedis covalently linked and one strand is nicked. According to thisembodiment, any of the ends can contain a type IA, type II, or type IBtopoisomerase recognition site, or can comprise a cleavage productthereof, provided that the first ds recombinant nucleotide moleculecontains a topoisomerase recognition site (e.g., a type IA or a type Htopoisomerase recognition site) at or near a 5′ terminus, or a cleavageproduct thereof, and only one topoisomerase or topoisomerase recognitionsite is present at the ends that are to be linked. For example, wherethe first nucleic acid molecule comprises a site-specific type IAtopoisomerase recognition site at or near each of the first end and thesecond end, the method further can include contacting the first nucleicacid molecule and the second nucleic acid molecule with at least a thirdnucleic acid molecule which has, or can be made to have, a first end anda second end, each end further comprising a 5′ terminus and a 3′terminus, under conditions such that the topoisomerase (e.g., a type IAor a type II topoisomerase) can covalently link the 5′ terminus of thefirst end of the first nucleic acid molecule with the 3′ terminus of thefirst end of the second nucleotide sequence, and the 5′ terminus of thesecond end of the first nucleic acid molecule with the 3′ terminus ofthe first end of the third nucleotide sequence. It will be recognizedthat other combinations of ends and topoisomerase recognition sites, orcleavage products thereof, can be used to perform such a method of theinvention. Once nucleic acid molecules are joined by the methodsdescribed above, the resulting molecules may then be used inrecombination reactions, such as those described elsewhere herein.

A method of the invention also can be performed by contacting a firstnucleic acid molecule and a second nucleic acid molecule with at least athird nucleic acid molecule, which comprises a first end and a secondend, each end further comprising a 5′ terminus and a 3′ terminus,wherein the third nucleic acid molecule comprises a type IBtopoisomerase recognition site at or near the 3′ terminus of said firstend, or said second end, or both said first end and said second end; andat least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) type IBtopoisomerase under conditions such that the type IB topoisomerase cancovalently link the 3′ terminus of the first end or second end of thethird nucleic acid molecule to the 5′ terminus of the first end orsecond end of the second nucleic acid molecule. In such a method, wherethe third nucleic acid molecule comprises a type IB topoisomeraserecognition site at or near the 3′ terminus of the first end, thecontacting can be performed under conditions such that the type IBtopoisomerase can covalently link the 3′ terminus of the first end ofthe third nucleic acid molecule to the 5′ terminus of the first end ofthe second nucleic acid molecule. It will be recognized that othercombinations of ends and topoisomerase recognition sites, or cleavageproducts thereof, can be used to perform such a method of the invention.Once nucleic acid molecules are joined by the methods described above,the resulting molecules may then be used in recombination reactions,such as those described elsewhere herein.

In another embodiment, a method for generating a ds recombinant nucleicacid molecule covalently linked in one strand can be performed bycontacting 1) a first nucleic acid molecule having a first end and asecond end, wherein the first nucleic acid molecule has a site-specifictopoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site) at or near the 5′ terminus of an end anda type BB topoisomerase recognition site at or near the 3′ terminus ofthe other end; 2) at least a second nucleic acid molecule that has, orcan be made to have, a first end and a second end; 3) at least one(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) site-specific topoisomerase(e.g., a type IA or a type II topoisomerase); and 4) at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) type BB topoisomerase underconditions such that all components are in contact and the at least onetopoisomerase can effect its activity. For example, the topoisomerase,for which a recognition site is at or near the 5′ terminus, can be atype IA topoisomerase such as E. coli topoisomerase I, E. colitopoisomerase III, or a eukaryotic topoisomerase III. Upon cleavage of anucleic acid molecule, the type IA topoisomerase preferably is stablybound to the 5′ terminus, and the type IB topoisomerase preferably isstably bound at the 3′ terminus. Preferably, upon cleavage by thetopoisomerases, the cleaved nucleic acid molecule comprises a 3′overhanging sequence and a 5′ overhanging sequence. The method canfurther include contacting the ds recombinant nucleic acid molecule witha DNA ligase, thereby generating a ds recombinant nucleic acid moleculecovalently linked in both strands. Once nucleic acid molecules arejoined by the methods described above, the resulting molecules may thenbe used in recombination reactions, such as those described elsewhereherein.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in one strand by contacting a first nucleic acid molecule, asecond nucleic acid molecule, and at least a third nucleic acidmolecule, can further include a step for amplifying the ds recombinantnucleic acid molecule, particularly the covalently linked strand. Theamplification can be carried out by contacting the ds recombinantnucleic acid molecule with an amplification reaction primer pair,wherein a first primer of the pair can bind selectively to thecovalently linked strand at or near one end of the first or secondnucleic acid molecule and prime an amplification reaction toward theother nucleic acid molecule to generate a first extension product thatis complementary to the covalently-linked strand; and the second primerof the pair can bind selectively to the first extension product,typically at or near the 3′ terminus, and, in the presence of the firstprimer, can generate an amplification product using the covalentlylinked strand and the extension product (or extension products derivedtherefrom) as templates. The method can be performed such that thetopoisomerase recognition site (e.g., a type IA or a type IBtopoisomerase recognition site) is at or near the first end of the firstnucleic acid molecule, and can further include contacting the dsrecombinant nucleic acid molecule with an amplification reaction primerpair, wherein a forward primer is capable of binding to a nucleotidesequence at or near the second end of the first nucleic acid moleculeand wherein a reverse primer is capable of binding to a nucleotidesequence complementary to at least a portion of the third nucleic acidmolecule; and amplifying the ds recombinant nucleic acid molecule. Thefirst nucleic acid molecule can include a coding region and the thirdnucleic acid molecule can include a regulatory element. Furthermore, theends being linked can contain complementary overhanging sequences. Oncenucleic acid molecules are joined by the methods described above, theresulting molecules may then be used in recombination reactions, such asthose described elsewhere herein.

Representative embodiments of the disclosed methods for generating a dsrecombinant nucleic acid molecule covalently linked in one strand and,optionally, comprising one or more recombination sites, are illustratedin FIGS. 11A-11F. In FIG. 11A, one of the nucleic acid molecules has atopoisomerase attached to the 5′ terminus of one end such that, whenthis molecule, which has a 3′ overhang, is contacted with a secondnucleic acid molecule having a substantially complementary 3′ overhang,under suitable conditions, the nucleotides comprising the 3′ overhangscan hybridize and the topoisomerases can catalyze ligation. FIG. 11Bshows a first nucleic acid molecule having topoisomerase moleculeslinked to the 5′ terminus and 3′ terminus of two different ends of onenucleotide sequence, and further shows linkage of the first nucleic acidmolecule to two other nucleotide sequences to generate a nucleic acidmolecule which has one strand without any nicks and another strand withtwo nicks. FIG. 11C shows a first nucleic acid molecule having atopoisomerase molecule linked to the 5′ terminus of one end and a secondnucleic acid molecule having a topoisomerase molecule linked to the 5′terminus of one end, and further shows linkage of the first and secondnucleic acid molecule to one other nucleotide sequence to generate anucleic acid molecule which has one strand without any nicks and anotherstrand with two nicks. In FIG. 11D, one of the nucleic acid molecules tobe linked has site-specific type IA topoisomerases attached to the 5′terminus of both ends such that, when the nucleotide sequences arecontacted the complementary 3′ overhangs can hybridize and thetopoisomerases catalyze ligation. FIG. 11E shows another example oflinking three nucleic acid molecules together, using one nucleic acidmolecule that is topoisomerase-charged with a type IA topoisomerase at a5′ terminus and another nucleic acid molecule that istopoisomerase-charged with a type IB topoisomerase at a 3′ terminus ofthe opposite strand to be linked, such that when the nucleotidesequences are contacted the complementary 3′ overhangs can hybridize andthe topoisomerases catalyze ligation. FIG. 11F illustrates anotherexample of linking three nucleic acid molecules together, in this caseusing one nucleic acid molecule that is topoisomerase-charged with atopoisomerase (e.g., a type IA or a type II topoisomerase) at a first 5′terminus and is charged with a topoisomerase at a second 5′-terminus ofthe opposite strand, such that when the nucleotide sequences arecontacted under suitable conditions, the complementary 3′ overhangs canhybridize and the topoisomerases catalyze ligation. Once nucleic acidmolecules are joined by the methods described above, the resultingmolecules may then be used in recombination reactions, such as thosedescribed elsewhere herein.

The examples set forth in FIGS. 11A-11F show the ends of the nucleicacid molecules opposite those being linked as having blunt ends, andshows the being linked as having 3′ overhanging sequences. However, thesubstrate nucleic acid molecules can have any ends and overhangs asdesired, including both ends being blunt and/or complementary, orcombinations thereof, such that the ends can be ligated to each other,for example, to form circular molecules or to other nucleic acidmolecules having an appropriate end. Thus, one or more of the blunt endsas shown in FIGS. 11A-11F can be substituted with a nucleotide sequencecomprising a 5′ overhang or a 3′ overhang, either of which canconstitute a single nucleotide such as a thymidine residue or multiplenucleotides (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, etc. nucleotides), whichcan be the same or different. In certain embodiments of the disclosedmethods, a first nucleic acid molecule contains a blunt end to belinked, and a second nucleic acid molecule contains an overhang at theend which is to be linked by a site-specific topoisomerase (e.g., a typeIA or a type IB topoisomerase), wherein the overhang includes a sequencecomplementary to that comprising the blunt end, thereby facilitatingstrand invasion as a means to properly position the ends for the linkingreaction.

As exemplified in FIGS. 11A-11C, the ds recombinant nucleic acidmolecule generated using the methods of this aspect of the inventioninclude those in which one strand (not both strands) is covalentlylinked at the ends to be linked (i.e. ds recombinant nucleic acidmolecules generated using these methods contain a nick at each positionwhere two ends were joined). These embodiments are particularlyadvantageous in that a polymerase can be used to replicate the dsrecombinant nucleic acid molecule by initially replicating thecovalently linked strand. For example, a thermostable polymerase such asa polymerase useful for performing an amplification reaction such as PCRcan be used to replicate the covalently strand, whereas the strandcontaining the nick does not provide a suitable template forreplication.

The present invention also provides methods of covalently ligating theends of two different nucleic acid molecules or two ends of the samenucleic acid molecule, such that the product generated is ligated inboth strands and, therefore, does not contain a nick. Representativeembodiments of this aspect of the invention are illustrated in FIG. 12.For example, in FIG. 12A, one of the nucleic acid molecules hastopoisomerase molecules attached to the 3′ terminus and the 5′ terminusof one end such that, when this molecule, which has a 5′ overhang, iscontacted with a second nucleic acid molecule having a substantiallycomplementary 5′ overhang, under suitable conditions, the nucleotidescomprising the 5′ overhangs can hybridize and the topoisomerases cancatalyze ligation of both strands of the nucleic acid molecules. In FIG.12B, each end of the nucleic acid molecules to be linked has atopoisomerase molecule attached to the 3′ terminus such that, when thenucleotide sequences are contacted under suitable conditions,nucleotides comprising the 5′ overhangs can hybridize and thetopoisomerases catalyze ligation (compare FIG. 12C, in which each of thenucleic acid molecules to be linked has a topoisomerase attached to the5′ termini of the ends to be linked). FIG. 12D illustrates linking threenucleic acid molecules together via a nucleic acid molecule that istopoisomerase-charged at both termini of both ends. Similarly to FIG.11, the examples set forth in FIGS. 12A-12D show the ends of the nucleicacid molecules that are not being linked as having blunt ends. Asdiscussed with respect to FIG. 11, however, the substrate nucleic acidmolecules utilized in methods as exemplified in FIG. 12 can have anyends as desired, including topoisomerase-charged ends, such that theends can be ligated to each other, for example, to form circularmolecules or to other nucleic acid molecules having an appropriate end,blunt ends, 5′ overhangs, 3′ overhangs, and the like, as desired. Oncenucleic acid molecules are joined by the methods described above, theresulting molecules may then be used in recombination reactions, such asthose described elsewhere herein.

A covalently bound topoisomerase, in addition to catalyzing a ligationreaction, also can catalyze the reverse reaction, for example,religation of the 3′ nucleotide of the recognition sequence, to whichthe type IB topoisomerase is linked through the phosphotyrosyl bond, andthe nucleotide sequence that, prior to cleavage, comprised the 3′terminus of the nucleic acid molecule, and which, following cleavage,contains a free 5′ hydroxy group. As such, methods have been developedfor using a type IB topoisomerase to produce recombinant nucleic acidmolecules. For example, cloning vectors containing a bound type IBtopoisomerase have been developed and are commercially available(Invitrogen Corporation, Carlsbad, Calif.). Such cloning vectors, whenlinearized, contain a covalently bound type IB topoisomerase at each 3′end (“topoisomerase charged”). Nucleotide sequences such as thosecomprising a cDNA library, or restriction fragments, or sheared genomicDNA sequences that are to be cloned into such a vector are treated, forexample, with a phosphatase to produce 5′ hydroxyl termini, then areadded to the linearized topoisomerase-charged vector under conditionsthat allow the topoisomerase to ligate the nucleotide sequences at the5′ terminus containing the hydroxyl group and the 3′ terminus of thevector that contains the covalently bound topoisomerase. A nucleotidesequence such as a PCR amplification product, which is generatedcontaining 5′ hydroxyl ends, can be cloned into a topoisomerase-chargedvector in a rapid joining reaction (approximately 5 minutes at roomtemperature). The rapid joining and broad temperature range inherent tothe topoisomerase joining reaction makes the use oftopoisomerase-charged vectors ideal for high throughput applications,which generally are performed using automated systems.

Type II topoisomerases have not generally been used for generatingrecombinant nucleic acid molecules or cloning procedures, whereas typeIB topoisomerases, as indicated above, are used in a variety ofprocedures. As disclosed herein, type IA topoisomerases can be used in avariety of procedures similar to those described for the type IBtopoisomerases. However, previously described methods of using type IBtopoisomerases to ligate two or more nucleotide sequences have sufferedfrom the disadvantage that the bound topoisomerase only effects thejoining of the 3′ end of the strand to which it is attached and a secondstrand containing a 5′ hydroxyl group. Since the topoisomerase cannotligate the complementary strands, the nucleic acid molecules that aregenerated contain nicks. While the presence of such nicks does notprevent the use of the recombinant molecules for transfection of a hostcells, as the nicks generally are resolved intracellularly, the presenceof such nicks in double stranded nucleic acid molecules significantlylimits direct use of the recombinant molecules. For example, a strand ofa nucleic acid molecule containing a nick cannot be amplified by PCRbecause the primer extension reaction terminates at the nick. Thus,nucleic acid constructs prepared using a topoisomerase according topreviously described methods generally must be further treated, forexample, with a DNA ligase, to obtain a ds recombinant nucleic acidmolecule that is covalently linked in both strands and, therefore,useful for subsequent manipulations such as PCR.

Previously described methods for preparing nucleic acid constructs alsogenerally required numerous steps, particularly where more than twonucleotide sequences are to be ligated, and even more so where thesequences must be ligated in a predetermined orientation. For example,the nucleotide sequences to be linked generally are ligated sequentiallyto produce intermediate constructs, each of which must be cloned,amplified in a host cell, isolated, and characterized. The constructscontaining the correct sequences then must be isolated in a sufficientquantity and form such that the next nucleotide sequence can be ligated,and the process of cloning, amplifying, isolating and characterizingperformed again to identify the proper construct. Clearly, as the numberof different nucleotide sequences to be joined increases, so do thenumber of essentially repetitive procedures that must be performed, thusresulting in an expensive, laborious and lengthy process.

As disclosed herein, an advantage of a method of the invention forgenerating a ds recombinant nucleic acid molecule covalently linked inboth strands is that there is no need to perform a separate ligationreaction in order to obtain a functional ds recombinant nucleic acidmolecule covalently linked in both strands (see FIGS. 8 and 12). Inaddition, a method of this aspect of the invention can be performed suchthat, where a number of different nucleic acid molecules are to becovalently linked in a predetermined orientation, there is norequirement that intermediate constructs be cloned, characterized andisolated before proceeding to a subsequent step (see Example 1.B). Assuch, the methods of this aspect of the invention provide a means togenerate a ds recombinant nucleic acid molecule covalently linked inboth strands much more quickly and at a substantially lower cost thanwas possible using previously known methods.

As an additional advantage, the generated ds recombinant nucleic acidmolecules covalently linked in both strands are in a form that can beused directly in further procedures, for example, particular proceduresinvolving extension of a primer such as a PCR amplification procedure,or other transcription or translation procedure, because the generatedconstruct does not contain nicks at the sites where the ds nucleotidessequences have been joined. As disclosed herein, a method of theinvention for generating a ds recombinant nucleic acid moleculecovalently linked in one strand, in certain embodiments, also isadvantageous in that the generated ds recombinant nucleic acid moleculesare in a form that can be used directly in further procedures, forexample, particular procedures involving extension of a primer such as aPCR amplification procedure, or other transcription or translationprocedure, because in certain embodiments, the generated ds recombinantnucleic acid molecule contains one strand that does not contain a nickat the sites where the ds nucleotides sequences were joined.

The term “nucleotide sequence” or “nucleic acid molecule” is used hereinto refer to a discrete nucleic acid molecule. When used as such, theterm “nucleotide sequence” is used merely for convenience such that thecomponents in a composition or used in a method of the invention can beclearly distinguished. Thus, reference is made, for example, to “nucleicacid molecules”, which, in a method of the invention, correspond to thereactants (substrates) used to produce a recombinant “nucleic acidmolecule” product.

Certain methods of the invention are exemplified generally herein withreference to the use of type IB topoisomerase such as the Vacciniatopoisomerase, or a type IA topoisomerase. However, it will berecognized that the methods also can be performed using a topoisomeraseother than that exemplified, merely by adjusting the componentsaccordingly. For example, as described in greater detail below, methodsare disclosed for incorporating a type IB topoisomerase recognition siteat one or both 3′ termini of a linear nucleic acid molecule using a PCRprimer comprising, at least in part, a nucleotide sequence complementaryto the topoisomerase recognition site. In comparison, a topoisomeraserecognition site for a type IA or, if desired, type II topoisomerase,can be incorporated into a nucleic acid molecule by using a PCR primerthat contains the recognition site.

Cleavage of a nucleic acid molecule by a site specific type IBtopoisomerase results in the generation of a 5′ overhanging sequence inthe strand complementary to and at the same end as that containing thecovalently bound topoisomerase. Furthermore, as disclosed herein, PCRprimers can be designed that can incorporate a type IB topoisomeraserecognition site into a nucleic acid molecule, and that further canproduce, upon cleavage of the nucleic acid molecule by thetopoisomerase, a 5′ overhanging sequence in the complementary strandthat has a defined and predetermined sequence. As such, the methods arereadily adaptable to generating a ds recombinant nucleic acid moleculehaving the component nucleic acid molecule operatively linked in apredetermined orientation. In view of the present disclosure, it will berecognized that PCR primers also can be designed such that a type IAtopoisomerase recognition site can be introduced into a nucleic acidmolecule, including a library of diverse sequences, and, if desired,such that upon cleavage by a site-specific topoisomerase, generates a 3′overhanging sequence.

A method of generating a ds recombinant nucleic acid molecule covalentlylinked in both strands, as disclosed herein, extends the previouslyknown methods by providing a topoisomerase at or near the terminus ofeach nucleic acid molecule to be covalently linked. For example, withrespect to a type IB topoisomerase, the method provides a topoisomeraserecognition site, or a cleavage product thereof (i.e., a covalentlybound type IB topoisomerase), at or near the 3′ terminus of each linearnucleic acid molecule to be linked. As used herein, the term“topoisomerase recognition site” means a defined nucleotide sequencethat is recognized and bound by a site specific topoisomerase. Forexample, the nucleotide sequence 5′-(C/T)CCTT-3′ is a topoisomeraserecognition site that is bound specifically by most poxvirustopoisomerases, including vaccinia virus DNA topoisomerase I, which thencan cleave the strand after the 3′-most thymidine of the recognitionsite to produce a nucleotide sequence comprising 5′-(C/T)CCTT-PO₄-TOPO,i.e., a complex of the topoisomerase covalently bound to the 3′phosphate through a tyrosine residue in the topoisomerase (see Shuman,J. Biol. Chem. 266:11372-11379, 1991; Sekiguchi and Shuman, Nucl. AcidsRes. 22:5360-5365, 1994; each of which is incorporated herein byreference; see, also, U.S. Pat. No. 5,766,891; PCT/US95/16099;PCT/US98/12372). In comparison, the nucleotide sequence 5′-GCAACTT-3′ isthe topoisomerase recognition site for type IA E. coli topoisomeraseIII.

Topoisomerase-charged nucleic acid molecules, including those containinga topoisomerase covalently attached to a 5′ terminus or 3′ terminus orboth, of one or both ends of the nucleic acid molecule, can be generatedby any of a number of methods. In some cases and under the appropriateconditions, type I topoisomerases can cleave a single strandednucleotide sequence. For example, a domain comprising the amino-terminal67 kDa domain of E. coli topoisomerase I, which is a type IAtopoisomerase, can cleave a single stranded nucleotide sequencecontaining the topoisomerase recognition site. Where conditions are suchthat the topoisomerases can cleave a single stranded nucleotidesequence, cleavage of a nucleic acid molecule containing topoisomeraserecognition sites at the 5′ and 3′ termini of one end of nucleic acidmolecule can be performed in parallel. Alternatively, where one or bothof the topoisomerases requires a nucleic acid molecule for recognitionand cleavage, the reactions are performed serially, wherein the moreterminal (distal) of the topoisomerase recognition sites is cleavedfirst, then the more internal (proximal) site, which remains in a doublestranded context, is cleaved. For example, a nucleic acid moleculecontaining an E. coli topoisomerase III recognition site at or near a 5′terminus of an end and a Vaccinia type IB topoisomerase recognition siteat or near the 3′ terminus of the same end, and wherein the type IBrecognition site is closer to the end than the type IA recognition site,the nucleic acid molecule can be incubated with the Vacciniatopoisomerase, to produce a type IB topoisomerase charged nucleic acidmolecule, then with the E. coli topoisomerase, to produce a nucleic acidmolecule having the type IA topoisomerase bound to the 5′ terminus andthe type IB topoisomerase bound to the 3′ terminus. Accordingly, theinvention includes methods for producing nucleic acid moleculecomprising a topoisomerase attached to one or both termini of at leastone end, and further provides such topoisomerase-charged nucleic acidmolecules.

As used herein, the term “cleavage product,” when used in reference to atopoisomerase recognition site, refers to a nucleotide sequence that hasbeen cleaved by a topoisomerase, generally at its recognition site, andcomprises a complex of the topoisomerase covalently bound, in the caseof type IA or type II topoisomerase, to the 5′ phosphate group of the 5′terminal nucleotide in the topoisomerase recognition site, or in thecase of a type IB topoisomerase to the 3′ phosphate group of the 3′terminal nucleotide in the topoisomerase recognition site. Such acomplex, which comprises a topoisomerase cleaved nucleic acid moleculehaving the topoisomerase covalently bound thereto, is referred to hereinas a “topoisomerase-activated” or a “topoisomerase-charged” nucleotidesequence. Topoisomerase-activated nucleic acid molecules can be used ina method of the invention, as can nucleic acid molecules that contain anuncleaved topoisomerase recognition site and a topoisomerase, whereinthe topoisomerase can cleave the nucleic acid molecule at therecognition site and become covalently bound thereto.

In one embodiment of a method of generating a ds recombinant nucleicacid molecule covalently linked in both strands, a topoisomeraserecognition site is present at or near the 3′ terminus of the end ofeach nucleotide sequence to be linked such that, in the presence of atype IB topoisomerase, each nucleotide sequence is cleaved to produce a3′ terminus, which contains the topoisomerase covalently bound thereto(see FIG. 8). The nucleotide sequences to be covalently linked also cancontain a 5′ hydroxy group at the same end as that containing thetopoisomerase recognition site, or a 5′ hydroxyl group can be generatedusing a phosphatase. Upon contact of such nucleotide sequences, the sitespecific topoisomerase can ligate each strand containing a 3′ phosphateto a respective 5′ hydroxyl group, thereby generating a ds recombinantnucleic acid molecule covalently linked in both strands, which can beproduced as a linear, circular, or positively or negatively supercoilednucleic acid molecule.

Preferably, the 5′ termini of the ends of the nucleotide sequences to belinked by a type IB topoisomerase according to a method of certainaspects of the invention contain complementary 5′ overhanging sequences,which can facilitate the initial association of the nucleotidesequences, including, if desired, in a predetermined directionalorientation. Alternatively, the 5′ termini of the ends of the nucleotidesequences to be linked by a type IB topoisomerase according to a methodof certain aspects of the invention contain complementary 5′ sequenceswherein one of the sequences contains a 5′ overhanging sequence and theother nucleotide sequence contains a complementary sequence at a bluntend of a 5′ terminus, to facilitate the initial association of thenucleotide sequences through strand invasion, including, if desired, ina predetermined directional orientation. The term “5′ overhang” or “5′overhanging sequence” is used herein to refer to a strand of a nucleicacid molecule that extends in a 5′ direction beyond the terminus of thecomplementary strand of the nucleic acid molecule. Conveniently, a 5′overhang can be produced as a result of site specific cleavage of anucleic acid molecule by a type IB topoisomerase (see Example 1).

Preferably, the 3′ termini of the ends of the nucleotide sequences to belinked by a type IA topoisomerase according to a method of certainaspects of the invention contain complementary 3′ overhanging sequences,which can facilitate the initial association of the nucleotidesequences, including, if desired, in a predetermined directionalorientation. Alternatively, the 3′ termini of the ends of the nucleotidesequences to be linked by a topoisomerase (e.g., a type IA or a type IItopoisomerase) according to a method of certain aspects of the inventioncontain complementary 3′ sequences wherein one of the sequences containsa 3′ overhanging sequence and the other nucleotide sequence contains acomplementary sequence at a blunt end of a 3′ terminus, to facilitatethe initial association of the nucleotide sequences through strandinvasion, including, if desired, in a predetermined directionalorientation. The term “3′ overhang” or “3′ overhanging sequence” is usedherein to refer to a strand of a nucleic acid molecule that extends in a3′ direction beyond the terminus of the complementary strand of thenucleic acid molecule. Conveniently, a 3′ overhang can be produced uponcleavage by a type IA or type II topoisomerase.

The 3′ or 5′ overhanging sequences can have any sequence, thoughgenerally the sequences are selected such that they allow ligation of apredetermined end of one nucleic acid molecule to a predetermined end ofa second nucleotide sequence according to a method of the invention(FIG. 9C, see, also Example 1.B). As such, while the 3′ or 5′ overhangscan be palindromic, they generally are not because nucleic acidmolecules having palindromic overhangs can associate with each other,thus reducing the yield of a ds recombinant nucleic acid moleculecovalently linked in both strands comprising two or more nucleic acidmolecules in a predetermined orientation. For example, the 5′overhanging sequences of nucleic acid molecules shown in FIG. 9A arepalindrome and, therefore, the association, for example, of a first CMVelement with a second CMV element through the AGCT overhang is just aslikely as the association of a CMV element with a GFP element throughthe AGCT overhang. As such, the efficiency of generating a constructcomprising an operatively covalently linked construct containing, inorder from 5′ to 3′, a CMV element, a GFP element and a BGH elementwould be reduced as compared to the efficiency of generating such aconstruct using the elements as shown in FIG. 9C. The elements shown inFIG. 9B contain palindromic overhangs at one end of the GFP element andat the end of the BGH element shown and, therefore, would be lessefficient than the elements of FIG. 9C, but more efficient than those inFIG. 9A, for generating the desired construct.

A nucleotide sequence used in the methods and kits of the currentinvention can be designed to contain a bridging phosphorothioate toprevent religation after topoisomerase-cleavage. For example, where thetopoisomerase is E. coli topoisomerase III, the bridgingphosphorothioate can be incorporated between the two thymidines of theGCAACTT cleavage/recognition sequence. When cleaved, the clippedsequence contains a 3′-SH instead of a 3′-OH, thus preventing religation(see Burgin, et al, Nucl. Acids Res. 23:2973-2979, 1995).

A nucleic acid molecule useful in a method or kit of an aspect of theinvention can be amplified by an amplification method such as PCR tocontain a topoisomerase recognition site at a 3′ or 5′ terminus of anend. Furthermore, one or both primers used for PCR can be designed suchthat, upon cleavage of an amplified nucleic acid molecule, the cleavednucleic acid molecule contains a 5′ or 3′ overhang at one or both ends.In one embodiment, PCR primers are designed such that the 5′ overhangingsequence on a first nucleic acid molecule is complementary to a 5′overhanging sequence on a second (or other) nucleic acid molecule,thereby facilitating the association of the nucleotide sequences,preferably in a predetermined orientation, whereupon they can becovalently linked according to a method of the invention. In accordancewith the invention, by designing unique overhanging sequences for thedifferent nucleic acid molecule to be linked, any number of nucleic acidmolecules can be linked in a desired order and/or orientation.

It should be recognized that PCR is used in two ways with respect to themethods of the invention. In one aspect, PCR primers are designed toimpart particular characteristics to a desired nucleic acid molecule,for example, a nucleic acid molecule that encodes a transcriptional ortranslational regulatory element or a coding sequence of interest suchas an epitope tag or cell compartmentalization domain. In this aspect,the PCR primers can be designed such that, upon amplification, thenucleic acid molecule contains a topoisomerase recognition site at oneor both ends, as desired. As disclosed herein, the PCR primer also caninclude an additional sequence such that, upon cleavage of theamplification product by a site specific topoisomerase, the cleavednucleic acid molecule contains a 5′ or 3′ overhanging sequence at thetopoisomerase cleaved end. In an embodiment of the invention involving atopoisomerase that binds and cleaves a 5′ terminus (e.g., an embodimentinvolving a type IA topoisomerase), the PCR primers can be designed tocontain a bridging phosphorothioate linkage (see above), which can blockreligation after topoisomerase cleavage and can assist in the generationof a topoisomerase charged amplification product.

Overhanging sequences generated using PCR can include a singlenucleotide overhang that is generated as an artifact of the PCRreaction. For example, a polymerase such at Taq, which does not have aproof-reading function and has an inherent terminal transferaseactivity, is commonly used, and produces PCR products containing asingle, non-template derived 3′ A overhang at each end. Theseamplification products can be linked to topoisomerase charged nucleicacid molecules containing a single 3′ T overhang or a single 3′ dUoverhang, which, for a T/A cloning reaction, can be a vector (see U.S.Pat. Nos. 5,487,993 and 5,856,144, each of which is incorporated hereinby reference), at one or both ends, using the methods of the invention.

PCR also is used to amplify a covalently linked ds recombinant nucleicacid molecule covalently linked in one or both strands, generated by amethod of the invention. For example, as illustrated in FIG. 13, amethod of the invention can generate an expressible ds recombinantnucleic acid molecule from three substrate nucleic acid moleculesincluding a nucleotide sequence comprising a promoter, a nucleotidesequence comprising a coding sequence, and a nucleotide sequencecomprising a polyadenylation signal. The generation of the dsrecombinant nucleic acid molecule can be facilitated by theincorporation of complementary 3′ (or 5′) overhanging sequences at theends of the ds nucleotides sequences to be joined. For example, theexpressible ds recombinant nucleic acid molecule can be generated bycontacting a first nucleic acid molecule having a type IA topoisomeraseat a 5′ terminus of a first end and a type IB topoisomerase at a 3′terminus of a second end with a second nucleic acid molecule and a thirddouble stranded nucleotide sequence. By designing a PCR primer paircontaining a first primer that is specific for a portion of thenucleotide sequence comprising the promoter that is upstream from thepromoter, and a second primer that is specific for a portion of thenucleotide sequence comprising the polyadenylation signal that is downstream of the signal, only a full length functional ds recombinantnucleic molecule containing the promoter, coding sequence andpolyadenylation signal in the correct (predetermined) orientation willbe amplified. In particular, partial reaction products, for example,containing only a promoter linked to the coding sequence, and reactionproducts containing nicks are not amplified. Thus, PCR can be used tospecifically design a nucleic acid molecule such that it is useful in amethod of the invention, and to selectively amplify only those reactionproducts having the desired components and characteristics.

As used herein, the term “covalently linked,” when used in reference toa ds recombinant nucleic acid molecule, means that the nucleic acidmolecule is generated from at least two nucleic acid molecules that areligated together, in both strands, by a topoisomerase mediated ligation.It should be recognized, for example, that a topoisomerase covalentlybound to one of the nucleic acid molecules to be covalently linked canbe the same as or different from the topoisomerase covalently bound tothe other nucleic acid molecule. Thus, a Vaccinia topoisomerase can becovalently bound to one nucleic acid molecule and another poxvirus oreukaryotic nuclear type IB topoisomerase can be bound to the otherstrand. Generally, however, the topoisomerases, where different, aremembers of the same family, for example, type IA or type IB or type II,although, where the topoisomerases are covalently bound, for example, toa 5′ phosphate and generate complementary 3′ overhangs, thetopoisomerase can be from different families, for example, type IA andtype II.

The term “covalently linked” also is used herein in reference to asingle stranded or double stranded nucleic acid molecule that isgenerated from at least two nucleotide sequences that are ligatedtogether in one strand. For example, a ds recombinant nucleic acidmolecule that is generated when a first topoisomerase-charged nucleicacid molecule that includes one topoisomerase bound at or near a 5′terminus contacts a second ds nucleotide sequence under conditions suchthat the topoisomerases can covalently link the 5′ terminus of the firstnucleic acid molecule to which it is bound, to the 3′ terminus of thesecond nucleic acid molecule, can generate a ds recombinant nucleic acidmolecule covalently linked in one strand.

In one embodiment, a ds recombinant nucleic acid molecule covalentlylinked in both strands generated according to a method of the inventiondoes not contain a nick in either strand at the site where twonucleotide sequences are ligated, although it can contain nickselsewhere in the molecule. In a method for generating a ds recombinantnucleic acid molecule covalently linked in one strand, a ds recombinantnucleic acid molecule is generated that contains a nick at least at theposition where ends were linked in the complementary strands. Thisnicked ds recombinant nucleic acid molecule can be converted to a dsrecombinant nucleic acid molecule covalently linked in both strands byintroducing the nicked ds recombinant nucleic acid molecule into a cell,or by subjecting the ds recombinant nucleic acid molecule to a ligationreaction, such as using a ligase, as is well known in the art.

The term “recombinant” is used herein to refer to a nucleic acidmolecule that is produced by linking at least two nucleotide sequencesaccording to a method of the invention. As such, a ds recombinantnucleic acid molecule encompassed within the present invention isdistinguishable from a nucleic acid molecule that may be produced innature, for example, during meiosis. For example, a ds recombinantnucleic acid molecule covalently linked in both strands generatedaccording to a method of certain aspects of the invention can beidentified by the presence of the two topoisomerase recognition sites,one present in each of the complementary strands, at or near the site atwhich the nucleic acid molecules were joined.

A method of the invention can be performed by contacting a first nucleicacid molecule having a first end and a second end, wherein at the firstend or second end or both, the first nucleic acid molecule has atopoisomerase recognition site, or cleavage product thereof, at or nearthe 3′ terminus and has (or can be made to have, for example, by contactwith a phosphatase) a hydroxyl group at the 5′ terminus of the same end;at least a second nucleic acid molecule having a first end and a secondend, wherein at the first end or second end or both, the at least secondnucleic acid molecule has a topoisomerase recognition site, or cleavageproduct thereof, at or near the 3′ terminus and has (or can be made tohave) a hydroxyl group at the 5′ terminus of the same end; and atopoisomerase, under conditions such that the components are in contactand the topoisomerase can effect its activity. Upon contact of thetopoisomerase with the first and second (or other) nucleic acidmolecules, and cleavage, where necessary, each nucleotide sequencecomprises at the cleavage site a covalently bound topoisomerase at the3′ terminus and has, or can have, a hydroxyl group at the 5′ terminussuch that, upon contact, the first and at least second nucleotidesequences are covalently linked in both strands. Accordingly, theinvention provides a ds recombinant nucleic acid molecule covalentlylinked in both strands produced by such a method.

As used herein, the term “at or near,” when used in reference to theproximity of a topoisomerase recognition site to the 3′ (type IB) or 5′(type IA or type II) terminus of a nucleotide sequence, means that thesite is within about 1 to 100 nucleotides from the 3′ terminus or 5′terminus, respectively, generally within about 1 to 20 nucleotides fromthe terminus, and particularly within about 2 to 12 nucleotides from therespective terminus. An advantage of positioning the topoisomeraserecognition site within about 10 to 15 nucleotides of a terminus isthat, upon cleavage by the topoisomerase, the portion of the sequencedownstream of the cleavage site can spontaneously dissociate from theremaining nucleotide sequence, which contains the covalently boundtopoisomerase (referred to generally as “suicide cleavage”; see, forexample, Shuman, supra, 1991; Andersen et al., supra, 1991). Where atopoisomerase recognition site is greater than about 12 to 15nucleotides from the terminus, the nucleotide sequence upstream ordownstream of the cleavage site can be induced to dissociate from theremainder of the sequence by modifying the reaction conditions, forexample, by providing an incubation step at a temperature above themelting temperature of the portion of the duplex including thetopoisomerase cleavage site.

An additional advantage of constructing a first or second (or other)nucleic acid molecule to comprise, for example, a type IB topoisomeraserecognition site about 2 to 15 nucleotides from one or both ends is thata 5′ overhang is generated following cleavage of the nucleic acidmolecule by a site specific topoisomerase. Such a 5′ overhangingsequence, which would contain 2 to 15 nucleotides, respectively, can bedesigned using a PCR method as disclosed herein to have any sequence asdesired. Thus, where a cleaved first nucleic acid molecule is to becovalently linked to a selected second (or other) nucleic acid moleculeaccording to a method of the invention, and where the selected sequencehas a 5′ overhanging sequence, the 5′ overhang on the first nucleic acidmolecule can be designed to be complementary to the 5′ overhang on theselected second (or other) ds sequence such that the two (or more)sequences are covalently linked in a predetermined orientation due tothe complementarity of the 5′ overhangs. As discussed above, similarmethods can be utilized with respect to 3′ overhanging sequencesgenerated upon cleavage by, for example, a type IA or type IItopoisomerase.

As used herein, reference to a nucleotide sequence having “a first end”and “a second end” means that the nucleotide sequence is linear. Asubstrate nucleic acid molecule can be linear or circular, includingsupercoiled, although, as a result of cleavage by one or moretopoisomerases, a linear topoisomerase-charged nucleic acid moleculegenerally is produced. For example, a circular nucleic acid moleculecontaining two type IB topoisomerase recognition sites within about 100nucleotides of each other and in the complementary strands, preferablywithin about twenty nucleotides of each other and in the complementarystrands, can be contacted with a site specific type IB topoisomerasesuch that each strand is cleaved and the intervening sequencedissociates, thereby generating a linear nucleic acid molecule having atopoisomrerase covalently bound to each end.

It should be recognized that reference to a first end or a second end ofa nucleic acid molecule is not intended to imply any particularorientation of the nucleotide sequence, and is not intended to imply arelative importance of the ends with respect to each other. Where anucleotide sequence having a first end and second end is a doublestranded nucleotide sequence, each end contains a 5′ terminus and a 3′terminus. Thus, reference is made herein, for example, to a nucleotidesequence containing a topoisomerase recognition site at a 3′ terminusand a hydroxyl group at the 5′ terminus of the same end, which can bethe first end or the second end.

A method of the invention can be performed using only a first nucleicacid molecule and a second nucleic acid molecule, or can additionallyinclude a third, fourth or more nucleic acid molecules as desired.Generally, each such nucleotide sequence contains a topoisomeraserecognition site, or a cleavage product thereof, at or near at least one3′ or 5′ terminus, and can contain a hydroxyl group at the 5′ terminusof the same end, or a hydroxyl group can be generated using aphosphatase. Where a nucleotide sequence does not contain atopoisomerase recognition site at or near an end to be linked to asecond nucleotide sequence, a topoisomerase recognition site can beintroduced into the nucleotide sequence using a method as disclosedherein, for example, by PCR amplification of the sequence using a primercomprising a complement of the topoisomerase recognition site.

The terms “first nucleotide sequence,” “second nucleotide sequence,”“third nucleotide sequence,” and the like, are used herein only toprovide a means to indicate which of several nucleotide sequences isbeing referred to. Thus, absent any specifically defined characteristicwith respect to a particular nucleotide sequence, the terms “first,”“second,” “third” and the like, when used in reference to a nucleotidesequence, or a population or plurality of nucleotide sequences, are notintended to indicate any particular order, importance or otherinformation about the nucleotide sequence. Thus, where an exemplifiedmethod refers, for example, to using PCR to amplify a first nucleic acidmolecule such that the amplification product contains a topoisomeraserecognition site at one or both ends, it will be recognized that,similarly, a second (or other) nucleic acid molecule also can be soamplified.

The term “at least a second nucleotide sequence” is used herein to meanone or more nucleotide sequences in addition to a first nucleotidesequence. Thus, the term can refer to only a second nucleotide sequence,or to a second nucleotide sequence and a third nucleotide sequence (ormore). As such, the term “second (or other) nucleotide sequence” or“second (and other) nucleotide sequences” is used herein in recognitionof the fact that the term “at least a second nucleotide sequence” canrefer to a second, third or more nucleotide sequences. It should berecognized that, unless indicated otherwise, a nucleotide sequenceencompassed within the meaning of the term “at least a second nucleotidesequence” can be the same or substantially the same as a firstnucleotide sequence. For example, a first and second nucleic acidmolecule can be the same except for having complementary 5′ overhangingsequences produced upon cleavage by a topoisomerase such that the firstand second nucleic acid molecules can be covalently linked using amethod of the invention. As such, a method of the invention can be usedto produce a concatenate of first and second nucleic acid molecules,which, optionally, can be interspersed, for example, by a third nucleicacid molecule such as a regulatory element, and can contain thecovalently linked sequences in a predetermined directional orientation,for example, each in a 5′ to 3′ orientation with respect to each other.

As disclosed herein, a method of the invention provides a means tocovalently link, two or more ds nucleotides in a predetermineddirectional orientation. The term “directional orientation” or“predetermined directional orientation” or “predetermined orientation”is used herein to refer to the covalent linkage, of two or morenucleotide sequences in a particular order. Thus, a method of theinvention provides a means, for example, to covalently link, a promoterregulatory element upstream of a coding sequence, and to covalently linka polyadenylation signal downstream of the coding region to generate afunctional expressible ds recombinant nucleic acid molecule; or tocovalently link two coding sequences such that they can be transcribedand translated in frame to produce a fusion polypeptide.

A method of the invention also can be performed by contacting a firstnucleic acid molecule having a first end and a second end, wherein atthe first end or second end or both, the first nucleic acid molecule hasa type IB topoisomerase covalently bound at the 3′ terminus(topoisomerase-charged) and has (or can be made to have) a hydroxylgroup at the 5′ terminus of the same end; and at least a second type IBtopoisomerase-charged nucleic acid molecule, which has (or can be madeto have) a hydroxyl group at the 5′ terminus at the same end. Uponcontact of the topoisomerase-activated first and at least secondnucleotide sequences at the ends containing the topoisomerase and a 5′hydroxyl group, phosphodiester bonds are formed in each strand, therebygenerating a ds recombinant nucleic acid molecule covalently linked inboth strands.

The invention further provides methods for linking two or more (e.g.,two, three, four, five, six, seven, etc.) nucleotide sequences, whereinthe linked ds recombinant nucleic acid molecule is covalently linked inone strand, but not both strands, (i.e. the ds recombinant nucleic acidmolecule contains a nick in one strand at each position where two endswere joined to generate the ds recombinant nucleic acid molecule).Further, one or more of the nucleotide sequences may comprise one ormore recombination sites. Using the schematic shown in FIG. 11A forpurposes of illustration, the invention includes methods for linking atleast two nucleotide sequences comprising contacting a first nucleicacid molecule having a first end and a second end, wherein at the firstend at the second end or at both ends, the first nucleic acid moleculehas a site-specific type IA topoisomerase covalently bound to the 5′termini; and a second nucleic acid molecule which does not havetopoisomerase covalently bound to either termini of at least one end.Further, the second nucleotide sequence will typically have hydroxylgroups at the 3′ termini of the end being joined to the first nucleicacid molecule. In many instances, the two nucleotide sequences to bejoined will have either 3′ or 5′ overhangs with sufficient sequencecomplementarity to allow for hybridization. In related embodiments, thefirst and second nucleic acid molecules described above may be first andsecond ends of the same nucleic acid molecule. Thus, connection of thetwo ends results in the formation of a circularized molecule. Oncenucleic acid molecules are joined by the methods described above, theresulting molecules may then be used in recombination reactions, such asthose described elsewhere herein. The invention further includes nucleicacid molecules prepared by methods of the invention, compositionscomprising such nucleic acid molecules, and methods for using suchnucleic acid molecules.

Using the schematic shown in FIG. 11B for purposes of illustration, theinvention includes methods for joining three or more nucleotidesequences. While any number of variations of the invention are possible,three nucleotide sequences may be joined by the use of a linker moleculewhich contains topoisomerases at or near both the 5′ and 3′ termini ofone strand, and optionally one or more recombination site. Thus, uponjoining of the three nucleotide sequences, a single nucleotide sequenceis formed which contains a first strand with no nicks at the junctionpoints, and a second strand with nicks at the junction points. Thisprocess has the advantage of employing a single topoisomerase modifiedmolecule to join three nucleotide sequences together. Once nucleic acidmolecules are joined by the methods described above, the resultingmolecules may then be used in recombination reactions, such as thosedescribed elsewhere herein. The invention further includes nucleic acidmolecules prepared by methods of the invention, compositions comprisingsuch nucleic acid molecules, and methods for using such nucleic acidmolecules.

The invention further provides methods for covalently linking bothstrands of two or more (e.g., two, three, four, five, six, seven, etc.)nucleic acid molecules. Using the schematic shown in FIG. 12A forpurposes of illustration, the invention includes methods for linking atleast two nucleotide sequences comprising contacting a first nucleicacid molecule having a first end and a second end, wherein at the firstend, at the second end, or at both ends, the first nucleic acid moleculehas two topoisomerases (e.g., a type IA and a type IB topoisomerase) oneeach covalently bound to the 3′ and 5′ termini; and a second nucleicacid molecule which does not have topoisomerase covalently bound toeither termini of at least one end. Further, the second nucleotidesequence will often have hydroxyl groups at the 51 and 3′ termini of theend being joined to the first nucleic acid molecule. In many instances,the two nucleotide sequences to be joined will have either 3′ or 5′overhangs with sufficient sequence complementarity to allow forhybridization, and, optionally, one or more recombination sites. Inrelated embodiments, the first and second nucleic acid molecules asdescribed above can be first and second ends of the same nucleic acidmolecule. Thus, connection of the two ends results in the formation of acircularized molecule. Once nucleic acid molecules are joined by themethods described above, the resulting molecules may then be used inrecombination reactions, such as those described elsewhere herein. Theinvention further includes nucleic acid molecules prepared by methods ofthe invention, compositions comprising such nucleic acid molecules, andmethods for using such nucleic acid molecules.

Using the schematic shown in FIG. 12D for purposes of illustration, theinvention includes methods for joining three or more nucleotidesequences. While any number of variations of the invention are possible,three nucleotide sequences may be joined by the use of a linker moleculewhich contains topoisomerases at or near both the 5′ and 3′ termini ofeach end and, optionally, one or more recombination sites. Thus, uponjoining of the three nucleotide sequences, a single nucleotide sequenceis formed which contains no nicks at the junction points. This processhas the advantage of employing a single topoisomerase modified moleculeto join three nucleotide sequences together. Once nucleic acid moleculesare joined by the methods described above, the resulting molecules maythen be used in recombination reactions, such as those describedelsewhere herein. The invention further includes nucleic acid moleculesprepared by methods of the invention, compositions comprising suchnucleic acid molecules, and methods for using such nucleic acidmolecules.

Substrates which particular reagents (e.g., enzymes) recognize and/orcatalyze reactions with can be used in methods of the invention toproduce nucleic acid molecules having particular characteristics. Forexample, reagents which catalyze nucleic acid modifications mayrecognize termini and/or generate termini having particular features.One example of such a feature is the presence or absence of a terminalphosphate group on the 3′ or 5′ strand. Such reagents, or combinationsof such reagents, may be used to prepare, for example, nucleic acidmolecules (1) from particular segments and/or (2) having a specific“pattern” of nicks (e.g., a nick in only one strand where two or moresegments are joined, nicks in alternating strands where three or moresegments are joined, etc.) or having no nicks in either strand.

Reagents (e.g., enzymes) which can be used in methods of the inventioninclude, but are not limited to, the following: ligases (e.g. DNA andRNA Ligases such as T4 DNA Ligase, T4 RNA ligase, E. coli DNA ligase,etc.), restriction enzymes (e.g., EcoRI, HpaII, BamHI, etc.), kinases(e.g., T4 polynucleotide kinase, etc.), phosphatases (e.g., calfintestinal alkaline phosphatase), topoisomerases, and polymerases (e.g.,proof-reading polymerases such as Pfu, Pfx, THERMALAcE™ (InvitrogenCorp., Carlsbad, Calif.), etc.), and non-proof-reading polymerases suchas Taq polymerase, Tfl polymerase, Tth polymerase, Thr polymerase,etc.).

The cleavage of nucleic acid molecules by many endonucleases (e.g.,restriction endonucleases) results in the formation of two new ends,wherein a hydroxyl group is present at the 3′ terminus of one end and aphosphate group is present at the 5′ terminus of the other end. Also,when exonucleases (e.g., snake venom phosphodiesterase, bovine spleenphosphodiesterase, E. coli exonuclease VII, lambda exonuclease, E. coliexonuclease III, etc.) digest nucleic acid molecules, they oftengenerate ends with (1) 5′ terminal hydroxyl groups and 3′ terminalphosphate groups or (2) 3′ terminal hydroxyl groups and 5′ terminalphosphate groups. Further, exonucleases typically digest only a singlestranded of a nucleic acid molecule but can use either single strandedand/or double stranded nucleic acids as substrates. In addition,exonucleases (e.g., exonucleases used in methods of the invention) maydigest nucleic acid molecules from the 3′ terminus, 5′ terminus, or boththe 3′ and 5′ termini. Also, kinases (e.g., T4 polynucleotide kinase,etc.) may be used to replace 5′ and/or 3′ terminal hydroxyl groups ofnucleic acid molecules with phosphate groups.

Many polymerases used for the amplification of nucleic acid molecules,for example, by PCR, generate nucleic acid products having 3′ terminalhydroxyl groups. In addition, the presence or absence of a phosphategroup, or other chemical group, at the 5′ terminus of a PCR product istypically determined by whether the primer used in the PCR reaction(s)contains a 5′ terminal phosphate or other chemical group. Thus, 5′terminal phosphate groups, hydroxyl groups, or other groups can beintroduced into PCR products by the use of primers which contain thesegroups at their 5′ termini. As a result, PCR can be used to generatenucleic acid molecules (i.e., the first nucleic acid molecule referredto below) which contain a desired arrangement of hydroxyl groups,phosphate groups and/or other groups on the 5′ and/or 3′ termini of oneor both ends of a linear nucleic acid molecule (e.g., 5′ phosphate groupand a 3′ hydroxyl group at one end and a 5′ hydroxyl group and a 3′hydroxyl group at the other end).

Each of the enzymes types listed above represents a general class oftools which can be used to generate nucleic acid molecules havingparticular characteristics (e.g., having a desired arrangement ofhydroxyl, phosphate and/or other groups on the 3′ and/or 5′ termini ofone or more ends). For example, double stranded, linear nucleic acidmolecules may be prepared in which the 5′ terminus and the 3′ terminusat one end each contain terminal hydroxyl groups and the 5′ terminus andthe 3′ terminus at the other end each contain terminal phosphate groups.Such ends may be prepared using the enzymes discussed above and/or otherreagents and methods known in the art.

Thus, the present invention contemplates the construction and use ofnucleic acid segments having particular characteristics (e.g., having adesired arrangement of hydroxyl, phosphate and/or other groups on the 3′and/or 5′ termini of one or more ends). Such nucleic acids include, butare not limited to, double-stranded, linear nucleic acid molecules whichhave first and second ends with the characteristics set out in Table 4.

TABLE 4 First End Second End 5′ Terminus 3′ Terminus 5′ Terminus 3′Terminus Phosphate Group Phosphate Group Phosphate Group Phosphate GroupPhosphate Group Phosphate Group Phosphate Group Hydroxyl Group PhosphateGroup Phosphate Group Hydroxyl Group Phosphate Group Phosphate GroupPhosphate Group Hydroxyl Group Hydroxyl Group Hydroxyl Group HydroxylGroup Phosphate Group Phosphate Group Hydroxyl Group Hydroxyl GroupPhosphate Group Hydroxyl Group Hydroxyl Group Hydroxyl Group HydroxylGroup Phosphate Group Hydroxyl Group Hydroxyl Group Hydroxyl GroupHydroxyl Group Hydroxyl Group Phosphate Group Phosphate Group PhosphateGroup Hydroxyl Group Phosphate Group Phosphate Group Hydroxyl GroupHydroxyl Group Phosphate Group Hydroxyl Group Phosphate Group HydroxylGroup Phosphate Group Hydroxyl Group Hydroxyl Group Phosphate GroupHydroxyl Group Phosphate Group Phosphate Group Phosphate Group HydroxylGroup Phosphate Group Hydroxyl Group Phosphate Group Hydroxyl GroupHydroxyl Group Phosphate Group Phosphate Group Hydroxyl Group HydroxylGroup Hydroxyl Group

Nucleic acid molecules having a desired arrangement of hydroxyl,phosphate and/or other groups on the 3′ and/or 5′ termini of one or moreends can be directionally linked to other nucleic acid molecules usinglinking reactions which require, for example, the presence of aparticular group on one or more termini of the molecule (e.g., either a5′ hydroxyl group or a 5′ phosphate group and/or a 3′ hydroxyl group ora 3′ phosphate group).

A number of reagents which catalyze the linkage of nucleic acid segmentsto each other will generally only recognize termini with particularchemical groups (e.g., a hydroxyl group or a phosphate group) present.For example, T4 DNA ligase will catalyze the ligation of the 3′ terminusof an end of a nucleic acid molecule to the 5′ terminus of a separateend of the same nucleic acid molecule or of a different nucleic acidmolecule, when the 5′ terminus contains a terminal phosphate group.Further, a number of topoisomerases (e.g., a type IB topoisomerases)will cleave and bind to the 3′ terminus of the end of a nucleic acidmolecule and catalyze the linkage of this 3′ terminus to the 5′ terminusof the end of the same nucleic acid molecule or of a different nucleicacid molecule, when the 5′ end contains a terminal hydroxyl group.Additionally, a number of topoisomerases (e.g. a type IA topoisomerases)will cleave and bind to the 5′ terminus of the end of a nucleic acidmolecule and catalyze the linkage of this 5′ terminus to the 3′ terminusof the end of the same nucleic acid molecule or of a different nucleicacid molecule, when the 3′ end contains a terminal hydroxyl group.

One example of such a linking reaction is where a first nucleic acidmolecule having a desired arrangement of groups on one or more termini(for example, a 5′ phosphate on one terminus and a 5′ hydroxyl on theother terminus) is linked to a second nucleic acid molecule thatcontains a type IB toposiomerase molecule covalently attached to aphosphate group at the 3′ terminus of only one end of the molecule,i.e., attached to the 3′ terminus of one strand of a double-strandednucleic acid molecule. In such an instance, the 3′ terminus of the endof the second nucleic acid molecule that contains the boundtoposiomerase can only be joined to the 5′ terminus of the end of thefirst nucleic acid molecule that contains the hydroxyl group. Thus,these two nucleic acid molecules can only be covalently linked in oneorientation.

A linear double stranded nucleic acid molecule which has phosphategroups at both of the 5′ and 3′ termini at both ends (see Table 4) maybe generated by any number of methods. One example of methods which maybe used to produce such molecules involves chemical synthesis of bothstrands of the double stranded nucleic acid molecule. These individualstrands may then be mixed under conditions which allow for the formationof the double stranded molecule.

Using reagents referred to above, as well as other reagents, nucleicacid molecules with various chemical groups at their termini can becovalently linked to each other in one or both strands. For example, afirst nucleic acid segment which contains a 5′ terminal phosphate groupand a 3′ terminal phosphate group with a type IB toposiomerase bound toit at one end may be linked in both strands to a second nucleic acidsegment which contains 5′ and 3′ terminal hydroxyl groups at one end. Inthis instance, the 3′ terminus of first nucleic acid segment whichcontains the toposiomerase molecule bound to it may be joined to the 5′terminus of the end of the second nucleic acid molecule. This linkingreaction may be catalyzed by the bound topoisomerase molecule. Further,the 5′ terminus of the same end of the first nucleic acid segments maybe covalently linked to the 3′ terminus of the end of the second nucleicacid segment to which it is joined by a ligase (e.g., T4 DNA ligase). Asa second example, a first nucleic acid segments is prepared with a“sticky end” (i.e., an overhang) generated by digestion with arestriction endonuclease that leaves a 5′ terminal phosphate grouppresent on the “sticky end”. The first nucleic acid segment is contactedwith a second nucleic acid segment which contains a compatible “stickyend” and a toposiomerase molecule bound to the 5′ terminus of this“sticky end”. The result is the covalent connection of these two nucleicacid segments in a single strand. Further, the nick in the other strandat the junction point may be sealed by the inclusion of a ligase, suchas T4 DNA ligase, in the reaction mixture.

Any number of variations of the above are possible depending on theavailable ends and the reagents used to prepare nucleic acid segmentswith ends for ligation by particular mechanisms or catalyzed byparticular reagents. One example of such a variation is where the 5′terminus of the “sticky end” of the first nucleic acid molecule referredcontains a hydroxyl group (e.g., the 5′ phosphate is removed by aphosphatase) and the second nucleic acid molecule contain a type IBtopoisomerase bound to the 3′ terminus of the compatible “sticky end”.

Thus, enzymes used to generate termini of nucleic acid molecules (e.g.,by amplification, by cleavage of a larger molecule, etc.) can beselected such that termini suitable for “downstream” reactions (e.g.,ligation reactions) may be generated. One example of such a process isshown in the upper portion of FIG. 41 and described as follows. Anucleic acid molecule may be amplified by PCR using a proofreadingpolymerase (e.g. Pfx, Pfu, etc.) which generates amplification productshaving predominantly blunt ends (i.e., neither terminus of theamplification product has an overhanging adenine or other residue) and3′ terminal hydroxyl groups at both ends. Blunt ended linkers whichcontain (1) nucleic acid of a T7 promoter and (2) a molecule of type IBtopoisomerase linked at or near the 3′ terminus of the end downstream ofthe promoter element (see Figure A). The 5′ terminus of the end of thelinker which contains the covalently bound topoisomerase contains aterminal phosphate. The result of the linking reaction, when conductedin the presence of T4 DNA ligase, is nucleic acid molecules which arecovalently linked in both strands at the junction point where the T7promoter element is joined to the PCR product. As one skilled in the artwould recognize, the process set out above and in FIG. 41 may beperformed with nucleic acid segments other than promoters and PCRproducts. In other words, essentially any nucleic acid segments may beused. Example of nucleic acid molecules which may be used in methods ofthe invention include those which have termini such as those set out inTable 4. Also, non-proof-reading polymerases (e.g., Taq polymerase) maybe used to generate the PCR product and the linkers containing the T7promoter element may have a “T” overhang for use in T/A cloning.

Further, the invention is not limited to methods for connecting twonucleic acid segments. Thus, the invention also includes methods forconnecting two or more nucleic acid segments to each other, wherein ateach connection point the nucleic acid segments are covalently linked toeach other in either one or both strands. The invention further includesnucleic acid molecules prepared by methods of the invention, as well ascompositions and reaction mixtures which contain the reaction productsand reaction precursors (e.g., nucleic acid segments which are to beconnected to each other by methods of the invention).

The process shown in FIG. 41 for the linkage of two nucleic acidsegments is non-directional. In other words, the two segments will beconnected to each other without regard to orientation. Methods of theinvention further include those directed to the selection, isolationand/or preparation of nucleic acid molecules which contain two or more(e.g., two, three, four, five, six eight, ten, etc.) nucleic acidmolecules connected in a particular order and/or orientation. Inperforming these methods, joining reactions may be designed, forexample, so that nucleic acid segments are connected to each other (1)in a particular order or orientation or (2) without regard toorientation and then assembled nucleic acid molecules which contain twoor more segments connected to each other in a particular order and/ororientation are selected and/or isolated.

One example of a method for performing the second process referred toimmediately above is shown in FIG. 41. The process shown in FIG. 41involves the connection of two nucleic acid molecules using methodsdescribed elsewhere herein, followed by the amplification of nucleicacid molecules which contain segments connected in a particularorientation. The amplification process employs primers (i.e., primers Aand B) which hybridize to different stands and at opposite ends of thelinkage product which is sought. Thus, when the T7 promoter is connectedto the PCR product in one orientation (e.g., the desired orientation),primers A and B hybridize to opposite strands and can be used to amplifythe nucleic acid molecule. However, when the T7 promoter is connected tothe PCR product in the other orientation (e.g., the non-desiredorientation), primers A and B hybridize to the same strand and can notbe used to amplify the nucleic acid molecule.

Thus, the invention includes, in part, methods for selectivelyamplifying nucleic acid molecules based on the order and/or orientationof nucleic acid segments which are joined by methods described elsewhereherein. In particular aspects, these methods involve performingamplification reactions in the presence of two or more primers whichhave been selected to amplify one or more desired nucleic acid moleculesassembled using methods described elsewhere herein. Nucleic acidmolecules selectively amplified by methods of the invention may beassembled by the joining of two or more nucleic acid segments. As oneskilled in the art would recognize, the selective amplification processdescribed above can be used to amplify nucleic acid molecules which areassembled from three, four, five, six, seven, etc. nucleic acidsegments. When three or more nucleic acid segments selectively amplifiedby methods described above, only those which contain the segmentscorresponding to the primers in the proper orientation will beamplified. Nucleic acid molecules which contain the correct segments andsegments in the proper order may be selected and/or isolated by the useof additional processes. For example, if nucleic acid segments 1, 2, and3 are connected to each other by methods of the invention, thenassembled nucleic acid segments containing nucleic acid segments 1 and 3in the desired orientation can be selectively amplified using primerscorresponding to sequences present in segments 1 and 3. Further,separation of nucleic acid molecules to obtain those which are of thesize represented by nucleic acid molecules comprising segments 1, 2, and3 may be performed to isolate these molecules. In such an instance,depending on how the nucleic acid segments are assembled, segment 2could be in either one particular orientation or in both orientationsAny number of such methods may be performed to obtain assembled nucleicacid molecules which contain nucleic acid segments connected to eachother in a desired orientation and/or order. The invention furtherincludes reaction mixtures and compositions for performing the methodsdescribed above, as well as nucleic acid molecules generated by thesemethods.

In the embodiment of the invention shown in FIG. 41, it is not necessaryto covalently link both strands at the junction between the linkercontaining the T7 Promoter element and the PCR product. After the firstround of amplification, both strands will be represented in thepopulation because, even if one strand contains a nick, the first roundof amplification will generate a full-length nucleic acid strandcorresponding to the nicked strand. Thus, both primers will hybridize tonucleic acid strands in the second and subsequent rounds ofamplification. As a result of the above, the T4 DNA ligase may beomitted from the methods schematically represented in FIG. 41.

Again using the process shown in FIG. 41 for reference, when a nucleicacid molecule is prepared as shown in the upper portion of FIG. 41, itmay be desirable to link both strands of the nucleic acid segments beingjoined when the product nucleic acid molecule is to be directly used(e.g., without one or more additional rounds of amplification) in aprocess such as transcription. This is so because strand separationoccurs during the transcription process and the presence of a nick inone of the strands often interferes with the transcription process.Thus, when nucleic acid molecules assembled as shown in FIG. 41 areintended for use for transcription, it will often be desirable togenerate nucleic acid molecules in which both strands are covalentlylinked at the junction between the nucleic acid segments. One exceptionto the above is where the template strand does not contain a nick afterlinking of the nucleic acid segments being joined. In other words, ininstances where the template strand is not nicked, transcription willefficiently occur even if a nick is present in the non-template strand.

The invention further provides methods for performing topoisomerasemediated joining reactions and recombination reactions which can beperformed in either a single tube or multiple tubes. For instance, allof the components necessary to perform both topoisomerase mediatedjoining reactions and recombination reactions can be combined in onetube and both reactions can occur essentially simultaneously. Examplesof topoisomerase/recombination reactions which can be performed ineither a single tube or in multiple tubes are shown in FIGS. 35-40.Thus, in particular embodiments, the invention provides single tubereactions in which (1) one or more nucleic acid molecules or two ends ofone nucleic acid molecule are linked to each other by a topoisomerasemediated reaction and (2) one or more recombination sites undergorecombination with one or more other recombination sites. Any number oftoposiomerase mediated joining reaction and/or recombination reactionsmay occur in processes of the invention. Further, these reactions mayoccur in any order. In particular embodiments, one or more nucleic acidmolecules in reaction mixtures of the invention will contain (1) one ormore recombination sites and (2) one or more topoisomerases or one ormore topoisomerase recognition sites.

As explained below in Example 9, in certain instances, topoisomeraseshave been found to inhibit particular recombination reactions. In suchinstances, nucleic acid molecules which have undergone toposiomerasemediated joining reaction(s) may be separated from topoisomerasespresent in the reaction mixture and then may used as substrates forrecombination reaction(s). Often in such instances, the topoisomerasemediated joining reaction(s) and the recombination reaction(s) willoccur in separate tubes. Examples of process by which products oftopoisomerase mediated joining reactions may be separated fromtopoisomerase include, but are not limited to, phenol/chloroformextraction, typically followed by precipitation of the nucleic acid(e.g., ethanol precipitation), and chromatography (e.g., columnchromatography).

Alternatively, topoisomerases present in the reaction mixture may beinactivated, for example, by heating (e.g., heating to about 65° C. forabout 60 min., about 70° C. for about 60 min., about 75° C. for about 60min., about 70° C. for about 40 min., about 75° C. for about 40 min.,about 80° C. for about 40 min., about 80° C. for about 30 min., about85° C. for about 20 min., about 90° C. for about 15 min., about 95° C.for about 5 min. or about 99° C. for about 1 min.) or by the use ofproteases (e.g., proteinase K). In this instance, it will generally bepossible for the topoisomerase mediated joining reaction(s) and therecombination reaction(s) to occur in the same tube.

In specific embodiments of single tube reactions, two or more nucleicacid segments, each comprising one or more topoisomerases ortoposiomerase recognition sites are joined to each other using atopoisomerase mediated joining reaction (e.g., a topoisomerase mediatedjoining reaction). After which, the tube is heated to about 85° C. forabout 20 min. and one or more recombinases are added. Further, if one ormore of the two or more nucleic acid segments do not compriserecombination sites or if recombination with additional nucleic acidsegments is desired, then nucleic acid segments which comprise one ormore recombination sites may be added. Typically, the recombinationsites present in the tube will be ones which are capable of recombiningwith each other.

In other specific embodiments of single tube reactions, two or morenucleic acid segments undergo recombination catalyzed by one or morerecombinases. After recombination has occurred, toposiomerase is thenadded to the tube to facilitate topoisomerase mediated joining ofnucleic acid segments. As above, additional nucleic acid segments may,optionally, be added to the reaction mixture along with thetopoisomerase. Further, when nucleic acid segments to which one or moretoposiomerases are attached are added to the reaction mixture, it willoften not be necessary to add additional topoisomerase. Thus, inparticular embodiments, topoisomerase modified nucleic segments may beadded to the above reaction mixtures and, depending on the particularreaction conditions, additional topoisomerase may or may not be added.

The invention also provides methods for preparing nucleic acid moleculeswhich contain one or more (e.g., one, two, three, four, five, six, etc.)multiple cloning sites. For example, one or more nucleic acid segmentsused in methods of the invention may comprise one or more multiplecloning sites. As another example, multiple cloning sites may be addedto nucleic acid segments used to prepare nucleic acid molecules bymethods of the invention or to nucleic acid molecules prepared bymethods of the invention by the attachment of linkers which contain oneor more multiple cloning sites. In related aspects, the inventionincludes nucleic acid molecules prepared by methods of the inventionwhich contain one or more multiple cloning sites, as well as the use ofone or more these multiple cloning sites to modify nucleic acidmolecules prepared by methods of the invention. The invention alsoprovides nucleic acid molecules produced by the methods described above,as well as uses of these molecules and compositions comprising thesemolecules.

Viral Vectors

The invention further provides methods for preparing nucleic acidmolecules having regions of viral nucleic acids, as well as nucleic acidmolecules prepared by such methods and compositions comprising thesenucleic acid molecules.

Adenoviruses are viral vectors that can be used, for example, in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia and the use of such vectors are includedwithin the scope of the invention. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993), present a reviewof adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994), demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationNos. WO94/12649 and WO 96/17053; U.S. Pat. No. 5,998,205; and Wang etal., Gene Therapy 2:775-783 (1995), the disclosures of all of which areincorporated herein by reference in their entireties.

Adeno-associated virus (AAV) and Herpes viruses, as well as vectorsprepared from these viruses have also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300;U.S. Pat. No. 5,436,146; Wagstaff et al., Gene Ther. 5:1566-70 (1998)).Herpes viral vectors are particularly useful for applications where geneexpression is desired in nerve cells.

The invention thus includes methods for preparing nucleic acid moleculeswhich have one or more functional properties of viral vectors (e.g.,adenoviral vectors, alphaviral vectors, herpes viral vectors,adeno-associated viral vectors, etc.). In particular embodiments,methods of the invention include the joining of nucleic acid segments,wherein one or more of the nucleic acid segments contains regions whichconfer upon product nucleic acid molecules the ability to function asviral vectors (e.g., the ability to replicate in specific host cells,the ability to be packaged into viral particles, etc.).

In particular embodiments, the invention includes methods for preparingadenoviral vectors by joining at least one (e.g., one, two, three, four,etc.) nucleic acid segment which comprises adenoviral sequences to oneor more other nucleic acid segments. Specific examples of adenoviralvectors, and nucleic acid segments which can be used to prepareadenoviral vectors are disclosed in U.S. Pat. Nos. 5,932,210, 6,136,594,and 6,303,362, the entire disclosures of which are incorporated hereinby reference. Adenoviral vector prepared by methods of the invention maybe replication competent or replication deficient.

One example of an adenoviral vector may be prepared by joining a nucleicacid segment comprising adenoviral nucleic acid to one or more othernucleic acid segments. For example, when a replication deficientadenoviral vector is desired, the adenoviral nucleic acid may havedeletions of all or part of one or more of the following regions: theE1a region, the E1b region, and/or the E3 region. Adenoviral vectorswhich contain deletions in these regions are described, for example, inU.S. Pat. No. 6,136,594. The invention further includes adenoviralvectors prepared by methods of the invention, as well as uses of thesevectors and compositions comprising these vectors. One example of a useof adenoviral vectors prepared by methods of the invention include thedelivery of nucleic acid segments to cells of a mammal (e.g., a human).Thus, the invention provides methods for preparing vector suitable foruse in gene therapy protocols. Typically, such vectors will bereplication deficient.

In specific embodiments, adenoviral vectors of the invention willcomprise substantially the entire adenoviral genome with the exceptionthat are deletions of all or part of one or more of the followingregions: the E1a region, the E1b region, and/or the E3 region. Infurther specific embodiments, non-adenoviral nucleic acid may be presentin one or more of the E1a region, the E1b region, and/or the E3 region.

In particular embodiments, adenoviral vectors prepared by methods of theinvention will contain at least one origin of replication and/or aselection marker which allows for amplification of the vector inprokaryotic cells, such as E. coli.

Adeno-associated viral vectors and Herpes viral vectors may be preparedby methods of the invention which are similar to those described above.Thus, the invention further provides methods for preparing such vectors,as well as vectors produced by these methods, uses of these vectors, andcompositions comprising these vectors.

The invention further provides methods for preparing alphaviral vectors(e.g., Sindbis virus vectors, Semliki Forest virus vectors, Ross Rivervirus vectors, Venezuelan equine encephalitis virus vectors, Westernequine encephalitis virus vectors, Eastern equine encephalitis virusvectors, etc.), as well as alphaviral vectors prepared by such methods,methods employing these alphaviral vectors and compositions comprisingthese alphaviral vectors.

In particular embodiments, the invention includes methods for preparingalphaviral vectors by joining at least one nucleic acid segment whichcomprises alphaviral sequences to one or more other nucleic acidsegments. Specific examples of alphaviral vectors and nucleic acidswhich can be used to prepare alphaviral vectors are described in U.S.Pat. Nos. 5,739,026 and 6,224,879, the GibcoBRL's Instruction Manual No.10179-018, “SFV Gene Expression System”, and Sindbis Expression Systemmanual (Invitrogen Corporation, Carlsbad, Calif.), catalog no. K750-01(version E), the entire disclosures of which are incorporated herein byreference.

In specific embodiments, alphaviral vector sequences used in methods ofthe invention to prepare alphaviral vectors will comprise one or more ofthe following components: one or more packaging signals (which may ormay not be of alphaviral origin), one or more subgenomic promoters,and/or nucleic acid encoding one or more non-structural protein (e.g.,nsp1, nsp2, nsp3, nsp4, etc.).

Alphaviral vectors of the invention may be introduced into cells as DNAor RNA molecules. When DNA forms of such vectors are introduced intocells, expression control sequences (e.g., inducible, repressible orconstitutive expression control sequences) may then be used to generateRNA molecules from which one or more non-structural proteins may betranslated. In specific embodiments, these non-structural proteins willform an RNA-dependent RNA polymerase which will amplify RNA moleculescorresponding to all or part of the transcript generated from the DNAform of the alphaviral vector. Thus, these non-structural proteins maycatalyze the production of additional copies of RNA molecules from RNAtemplates, resulting in RNA amplification. Further, a nucleic acidsegment for which high levels of expression is desired may be operablylinked to a subgenomic promoter, thus resulting in the production ofhigh levels of RNA corresponding to the nucleic acid segment.

In one exemplary embodiment, alphaviral vectors prepared by methods ofthe invention comprise DNA wherein an inducible promoter directstranscription of an RNA molecule which encodes nsp1, nsp2, nsp3, andnsp4 of a Sindbis virus and a Sindbis subgenomic promoter operativelylinked to a nucleic acid segment which is not of Sindbis viral origin.The invention also provides alphaviral vectors prepared by methods ofthe invention, methods of using such alphaviral vectors, andcompositions comprising such alphaviral vectors.

The invention further provides methods for joining nucleic acid segmentswherein one or more of the nucleic acid segments contains one or more(e.g., one, two, three, four, etc.) viral packaging signal (e.g., one ormore packaging signal derived from a virus referred to above). Thesepackaging signals can be used to direct the packaging of nucleic acidmolecules prepared by methods of the invention. One method for preparingpackaged nucleic acid molecules is by the introduction or expression ofnucleic acid molecules of the invention into packaging cell lines whichexpress proteins suitable for the production of virus-like particles.The invention further includes packaged nucleic acid molecules of theinvention, methods for preparing packaged nucleic acid molecules of theinvention, and compositions comprising packaged nucleic acid moleculesof the invention.

The present invention also provides compositions, and kits containingsuch compositions, including kits containing component useful forperforming methods of the invention. In one aspect, a composition of theinvention comprises isolated components characteristic of a step of amethod of the invention. For example, a composition of the invention cancomprise two or more of the same or different topoisomerase-chargednucleic acid molecules. As used herein, the term “different,” when usedin reference to the nucleic acid molecules of a composition of theinvention, means that the nucleic acid molecules share less than 95%sequence identity with each when optimally aligned, generally less than90% sequence identity, and usually less than 70% sequence identity.Thus, nucleic acid molecules that, for example, differ only in beingpolymorphic variants of each other or that merely contain different 5′or 3′ overhanging sequences are not considered to be “different” forpurposes of a composition of the invention. In comparison, differentnucleic acid molecules are exemplified by a first sequence encoding apolypeptide and second sequence comprising a regulatory element, or afirst sequence encoding a first polypeptide a second sequence encoding anon-homologous polypeptide.

Where a composition of the invention comprises more than two differentisolated nucleic acid molecules or more than two differenttopoisomerase-charged nucleic acid molecules, each of the nucleic acidmolecules is different from each other, i.e., they are all differentfrom each other. However, it will be recognized that each of the nucleicacid molecules, for example, a sequence referred to as a first nucleicacid molecule, generally comprises a population of such nucleotidesequences, which are identical or substantially identical to each other.Thus, it should be clear that the term “different” is used in comparing,for example, a first (or population of first) nucleic acid moleculeswith a second (and other) nucleic acid molecule. A compositioncomprising two or more different topoisomerase-charged nucleic acidmolecules can further comprise a topoisomerase. Examples of such nucleicacid molecules comprising the components of a composition of theinvention are disclosed herein and include, for example, codingsequences, transcriptional regulatory element, translational regulatoryelements, elements encoding a detectable or selectable markers such asan epitope tag or an antibiotic resistance gene, elements encodingpolypeptide domains such as cell compartmentalization domains or signalpeptides, and the like.

As used herein, the term “isolated” means that a molecule being referredto is in a form other than that in which it exists in nature. Ingeneral, an isolated nucleotide sequence, for example, can be anynucleotide sequence that is not part of a genome in a cell, or isseparated physically from a cell that normally contains the nucleotidesequence. It should be recognized that various compositions of theinvention comprise a mixture of isolated nucleic acid molecules. Assuch, it will be understood that the term “isolated” only is used inrespect to the isolation of the molecule from its natural state, butdoes not indicate that the molecule is an only constituent.

A composition of the invention can comprise two different nucleic acidmolecules, each of which contains a topoisomerase recognition site at ornear one or both ends, and a site specific topoisomerase, which can bindto and cleave the nucleic acid molecules at the topoisomeraserecognition site. Optionally, at least one of the different nucleic acidmolecules can be a topoisomerase-charged nucleic acid molecule.Preferably, the topoisomerase covalently bound to thetopoisomerase-charge nucleic acid molecule is of the same family as thetopoisomerase in the composition.

Various combinations of components can be used in a method of theinvention. For example, the method can be performed by contacting atopoisomerase-activated first nucleic acid molecule, which optionallycomprises one or more recombination sites; a second nucleic acidmolecule having a first end and a second end, wherein at the first endor second end or both, the second nucleotide sequence has atopoisomerase recognition site at or near the 3′ terminus, and ahydroxyl group at the 5′ terminus of the same end; and a topoisomerase.Where the 5′ terminus of one or both ends to be linked has a 5′phosphate group, a phosphatase also can be contacted with the componentsof the reaction mixture. Upon such contacting, the topoisomerase cancleave the second nucleotide sequence to produce atopoisomerase-activated second nucleic acid molecule, the phosphatase,if necessary, can generate a 5′ hydroxyl group at the same end, and thesecond nucleic acid molecule then can be covalently linked to thetopoisomerase-activated first nucleic acid molecule. As such, it will berecognized that a composition of the invention can comprise any ofvarious combinations of components useful for performing a method of theinvention. Once nucleic acid molecules are joined by the methodsdescribed above, the resulting molecules may then be used inrecombination reactions, such as those described elsewhere herein. Theinvention further includes nucleic acid molecules prepared by methods ofthe invention, compositions comprising such nucleic acid molecules, andmethods for using such nucleic acid molecules.

In general, a method of the invention for generating a ds recombinantnucleic acid molecule covalently linked in both strands is based on thedetermination that a ds recombinant nucleic acid molecule covalentlylinked in both strands can be produced by contacting a first nucleicacid molecule with a second nucleic acid molecule, wherein the first andsecond sequences each have, at the ends to be linked, a topoisomeraserecognition site, for example, 5′-(C/T)CCTT-3′ (Shuman, supra, 1991;U.S. Pat. No. 5,766,891). Upon cleavage, the site specific topoisomeraseis covalently bound at the 3′ terminus. Where the cleaved nucleotidesequences also contain a 5′ hydroxy group at the same end as the boundtopoisomerase, and the ends of the two nucleotide sequences associate,the topoisomerase on each 3′ terminus can covalently link that terminusto a 5′ hydroxyl group on the associated nucleotide sequence (see FIG.12B).

As used herein, reference to contacting a first nucleotide sequence andat least a second nucleotide sequence “under conditions such that allcomponents are in contact” means that the reaction conditions areappropriate for the topoisomerase-cleaved ends of the nucleotidesequences to come into sufficient proximity such that a topoisomerasecan effect its enzymatic activity and covalently link the 3′ or 5′terminus of a first nucleotide sequence to a 5′ or 3′ terminus,respectively, of a second nucleotide sequence. Examples of suchconditions, which include the reaction temperature, ionic strength, pH,and the like, are disclosed herein, and other appropriate conditions asrequired, for example, for particular 5′ overhanging sequences of thetermini generated upon topoisomerase cleavage, can be determinedempirically or using formulas that predict conditions for specifichybridization of nucleotide sequences, as is well known in the art (see,for example, (Sambrook et al., Molecular Cloning: A laboratory manual(Cold Spring Harbor Laboratory Press 1989); Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1987, and supplements through 1995), each of which is incorporatedherein by reference).

In one embodiment, a method of the invention provides a means to renderan open reading from a cDNA or an isolated genomic DNA sequenceexpressible by operatively linking one or more regulatory elements tothe putative coding sequence. Accordingly, a first nucleic acid moleculecomprising an open reading frame can be amplified by PCR using a primerpair that generates an amplified first nucleic acid molecule having atopoisomerase recognition site at one or both ends and, optionally, oneor more recombination sites, as desired, such that, upon cleavage by thesite specific topoisomerase, one or both ends contains a defined 5′ or3′ overhang or is blunt. Where both ends of the amplified first nucleicacid molecule are so constructed, the 5′ or 3′ overhanging sequencesgenerally, but not necessarily, are different from each other. Theamplified first nucleic acid molecule then can be contacted with asecond nucleic acid molecule comprising a desired regulatory elementsuch as a promoter and, in certain embodiments, (a) one or moretopoisomerase recognition sites, and with a topoisomerase and/or (b) oneor more recombination sites, under conditions which facilitaterecombination, such that the second nucleotide sequence is operativelycovalently linked to the 5′ end of the coding sequence according to amethod of the invention.

In such a method, a second (or other) nucleic acid molecule also cancomprise two or more regulatory elements, for example, a promoter, aninternal ribosome entry site and an ATG initiator methionine codon, orthe like, or other sequence of interest, for example, an sequenceencoding an epitope tag, in operative linkage with each other, and whichcan be operatively covalently linked to the 5′ end of a first nucleicacid molecule comprising a coding sequence. Such a method can furtherinclude contacting a third nucleic acid molecule comprising, forexample, a polyadenylation signal, which can be operatively covalentlylinked according to a method of the invention to the 3′ end of thecoding sequence, thereby generating an expressible ds recombinantnucleic acid molecule. As such, a method of the invention provides ameans for generating a functional ds recombinant nucleic acid moleculethat can be transcribed, translated, or both as a functional unit. Asdisclosed herein, the inclusion of complementary 5′ or 3′ overhangingsequences generated by topoisomerase cleavage at the termini of thenucleic acid molecules to be linked together by the site specifictopoisomerase facilitates the generation of a ds recombinant nucleicacid molecule having a desired directional orientation of the nucleotidesequences in the construct.

In another embodiment, a method of the invention is performed such thatthe first nucleic acid molecule or a second (or other) nucleic acidmolecule, or combination thereof, is one of a plurality of nucleotidesequences. As used herein, the term “plurality,” when used in referenceto a first or at least a second nucleotide sequence, means that thenucleotide sequences are related but different. For purposes of thepresent invention, the nucleotide sequences of a plurality are “related”in that each nucleotide sequence in the plurality contains at least atopoisomerase recognition site, or a cleaved form thereof, at one ormore termini and/or at least one recombination site. Furthermore, thenucleotide sequences of a plurality are “different” in that they cancomprise, for example, a cDNA library, a combinatorial library ofnucleotide sequences, a variegated population of nucleotide sequences,or the like. Methods of making cDNA libraries, combinatorial libraries,libraries comprising variegated populations of nucleotide sequences, andthe like are well known in the art (see, for example, U.S. Pat. No.5,837,500; U.S. Pat. No. 5,622,699; U.S. Pat. No. 5,206,347; Scott andSmith, Science 249:386-390, 1992; Markland et al., Gene 109:13-19, 1991;O'Connell et al., Proc. Natl. Acad. Sci., USA 93:5883-5887, 1996; Tuerkand Gold, Science 249:505-510, 1990; Gold et al., Ann. Rev. Biochem.64:763-797, 1995; each of which is incorporated herein by reference).

The present invention further provides a method of generating a dsrecombinant nucleic acid molecule covalently linked in both strands byamplifying a portion of a first nucleotide sequence using a PCR primerpair, wherein at least one primer of the primer pair encodes atopoisomerase recognition site or a complement thereof and, optionally,one or more recombination sites, thereby producing a first nucleic acidmolecule having a first end and a second end, wherein the first end orsecond end or both has a topoisomerase recognition site at the 3′terminus and/or the 5′ terminus; and contacting the first nucleic acidmolecule with at least a second nucleic acid molecule having a first endand a second end, wherein the first end or second end or both has atopoisomerase recognition site at the 3′ terminus and/or the 5′terminus, or a cleavage product thereof; and a topoisomerase (see FIG.12). When contacted under conditions such that an end of the firstnucleic acid molecule having a topoisomerase recognition site and an endof the at least second nucleic acid molecule having a topoisomeraserecognition site can associate, a ds recombinant nucleic acid moleculecovalently linked in both strands is generated. Once nucleic acidmolecules are joined by the methods described above, the resultingmolecules may then be used in recombination reactions, such as thosedescribed elsewhere herein. The invention further includes nucleic acidmolecules prepared by methods of the invention, compositions comprisingsuch nucleic acid molecules, and methods for using such nucleic acidmolecules.

As disclosed herein, a PCR method using primers designed to incorporateone or more topoisomerase recognition sites and, optionally, one or morerecombination sites at one or both ends of an amplified nucleic acidmolecule provides a convenient means for producing nucleic acidmolecules useful in a method of the invention. In certain embodiments,at least one of the primers of a primer pair is designed such that itcomprises, in a 5′ to 3′ orientation, a nucleotide sequencecomplementary to a topoisomerase recognition site, such that PCRintroduces a functional recognition site in the opposite strand (seeprimer sequences in FIG. 9D), and a nucleotide sequence complementary tothe 3′ end of a target nucleic acid molecule to be amplified (i.e., atarget specific region). In addition, the primer can contain, in aposition 5′ to the complement of the topoisomerase recognition site, adesired nucleotide sequence of any length (generally about 1 to 100nucleotide, usually about 2 to 20 nucleotides, and particularly about 4to 12 nucleotides), which, upon cleavage of the amplification product bya site specific topoisomerase, forms a desired 5′ overhang. The secondprimer of the PCR primer pair can be complementary to a desired sequenceof the nucleotide sequence to be amplified, and can comprise acomplement to a topoisomerase recognition site, a sequence that wouldgenerate a 5′ overhang upon cleavage by a site specific topoisomerase,or any other sequence, as desired.

Such a primer can comprise or encode any other sequence of interest,including, for example, a site specific integration recognition sitesuch as an att site, a lox site, or the like, or, as discussed above,can simply be used to introduce a topoisomerase recognition site into anucleic acid molecule comprising such a sequence of interest. A dsrecombinant nucleic acid molecule generated according to a method of theinvention and containing a site specific integration recognition sitesuch as an att site or lox site can be integrated specifically into adesired locus such as into a vector, a gene locus, or the like, thatcontains the required integration site, for example, an att site or loxsite, respectively, and upon contact with the appropriate enzymesrequired for the site specific event, for example, lambda Int and IHFproteins or Cre recombinase, respectively. The incorporation, forexample, of attB or attP sequences into a ds recombinant nucleic acidmolecule covalently linked in both strands according to a method of theinvention allows for the convenient manipulation of the nucleic acidmolecule using the GATEWAY™ Cloning System (Invitrogen Corporation,Carlsbad, Calif.).

In one embodiment, a construct generated according to a method of theinvention is further amplified by a PCR reaction or other amplificationreaction. Direct PCR of a ds recombinant nucleic acid molecule generatedaccording to a method of the invention is possible because the constructis covalently linked in at least one strand. As such, PCR can be used togenerate a large amount of the construct. More importantly, as indicatedabove, PCR provides an in vitro selection method for obtaining only adesired product generated according to a method of the invention,without obtaining partial reaction products. For example, a method ofthe invention can be used to generate a ds recombinant nucleic acidmolecule covalently linked in both strands comprising, operativelylinked in a 5′ to 3′ orientation, a first nucleic acid moleculecomprising a promoter, a second nucleic acid molecule comprising acoding region, and a third nucleic acid molecule comprising apolyadenylation signal.

As disclosed herein, a construct having a predetermined orientation canbe generated by including complementary 5′ overhanging sequences on theends of the nucleic acid molecules to be joined. By selecting a PCRprimer pair including a first primer complementary to the first nucleicacid molecule and upstream of the promoter sequence, and a second primercomplementary to the third nucleic acid molecule and downstream of thepolyadenylation signal, a functional amplification product comprisingthe promoter, coding region and polyadenylation signal can be generated.In contrast, partial reaction products that lack either the firstnucleic acid molecule or third ds nucleotide is not amplified becauseeither the first or second primer, respectively, would not hybridize tothe partial product. In addition, a construct lacking the second nucleicacid molecule would not be generated due to the lack of complementarityof the 5′ overhanging sequences of the first and third nucleic acidmolecules. As such, a method of the invention provides a means to obtaina desired functional ds recombinant nucleic acid molecule covalentlylinked in both strands.

The use of PCR in such a manner further provides a means to screen alarge number of nucleic acid molecules generated according to a methodof the invention in order to identify constructs of interest. Sincemethods for utilizing PCR in automated high throughput analyses areroutine and well known, it will be recognized that the methods of theinvention can be readily adapted to use in a high throughput system.Using such a system, a large number of constructs can be screened inparallel, and partial or incomplete reaction products can be identifiedand disposed of, thereby preventing a waste of time and expense thatwould otherwise be required to characterize the constructs or examinethe functionality of the constructs in further studies.

The methods of the invention have broad application to the field ofmolecular biology. As discussed in greater detail below, the methods ofthe invention can be used, for example, to label DNA or RNA probes, toperform directional cloning (see Example 1.B), to generate sense orantisense RNA molecules (see Example 2.A), to prepare bait or preyconstructs for performing a two hybrid assay (see Example 2.C), toprepare linear expression elements (see Examples 2.A and 2.B), and toprepare constructs useful for coupled in vitro transcription/translationassays (see Example 2.B). For example, a method of generating dsrecombinant nucleic acid molecules covalently linked in both strandsprovides a means to generate linear expression elements (LEEs), whichconsist of a linear nucleic acid molecule comprising two or morenucleotide sequences such as a promoter or other regulatory elementlinked to an open reading frame (see Example 1). LEEs have been reportedto efficiently transfect cells, thus bypassing a requirement for cloningthe expression element in a vector (Sykes and Johnston, Nat. Biotechnol.17:355-359, 1999). The components of a LEE can be noncovalently linked,or can be covalently linked via a ligation reaction. The preparation ofnoncovalently linked LEEs requires using PCR primers containingdeoxyuridine residues to amplify each nucleotide sequence component,then treating the PCR products with uracil-DNA glycosylase to generateoverhanging ends that can hybridize. However, the efficiency oftransfection using such noncovalently linked LEEs is variable, and, insome cases, much lower than the efficiency of covalently linked LEEs(Sykes and Johnston, supra, 1999). Furthermore, such LEEs are notsuitable for use as templates for PCR amplification because the primerextension reaction cannot proceed past nicks in the template and,therefore, is terminated producing incomplete reaction products.

A method of the invention provides a straightforward and simple means togenerate covalently linked LEEs, thereby avoiding the inconvenient andadditional steps previously described for preparing a LEE, as well asreducing variability in transfection efficiency as observed usingnoncovalently linked LEEs. For example, a first nucleic acid molecule,which encodes an open reading frame of interest, can be amplified by PCRas disclosed herein to contain a topoisomerase recognition site, orcleavage product thereof, on one or both ends. Furthermore, the PCRprimers can be designed such that, upon cleavage of the amplified firstnucleic acid molecule by a site specific topoisomerase, the cleavageproduct contains a predetermined and desired 5′ overhanging sequence. Asecond nucleotide sequence (and a third or more, as desired), inaddition to containing a topoisomerase recognition site, or cleavageproduct thereof, can include or encode a regulatory element, forexample, a promoter, an enhancer, a silencer, a splice acceptor site, atranslation start site, a ribosome recognition site or internal ribosomeentry site, a polyadenylation signal, an initiator methionine codon, ora STOP codon, or can encode any other desired sequence such as anepitope tag or cell compartmentalization domain. Preferably, the second(or other) nucleic acid molecule to be covalently linked to the firstnucleic acid molecule has a 5′ overhanging sequence that iscomplementary to the 5′ overhang at the end of the first nucleic acidmolecule to which it is to be linked. Upon contact of such nucleotidesequences in presence of a topoisomerase a promoter, for example, can beoperatively covalently linked to the 5′ terminus of the open readingframe, and a polyadenylation signal can be operatively covalently linkedto the 3′ terminus of the open reading frame, thereby generating acovalently linked functional LEE (see Example 1).

Examples of regulatory elements useful in the present invention aredisclosed herein and include transcriptional regulatory elements,translational regulatory elements, elements that facilitate thetransport or localization of a nucleotide sequence or polypeptide in (orout of) a cell, elements that confer a detectable phenotype, and thelike. Transcriptional regulatory elements include, for example,promoters such as those from cytomegalovirus, Moloney leukemia virus,and herpes virus, as well as those from the genes encodingmetallothionein, skeletal actin, phosphoenolpyruvate carboxylase,phosphoglycerate, dihydrofolate reductase, and thymidine kinase, as wellas promoters from viral long terminal repeats (LTRs) such as Roussarcoma virus LTR and operators; enhancers, which can be constitutivelyactive such as an immunoglobulin enhancer, or inducible such as SV40enhancer; and the like. For example, a metallothionein promoter is aconstitutively active promoter that also can be induced to a higherlevel of expression upon exposure to a metal ion such as copper, nickelor cadmium ion. In comparison, a tetracycline (tet) inducible promoteris an example of a promoter that is induced upon exposure totetracycline, or a tetracycline analog, but otherwise is inactive. Atranscriptional regulatory element also can be a tissue specificregulatory element, for example, a muscle cell specific regulatoryelement, such that expression of an encoded product is restricted to themuscle cells in an individual, or to muscle cells in a mixed populationof cells in culture, for example, an organ culture. Muscle cell specificregulatory elements including, for example, the muscle creatine kinasepromoter (Sternberg et al., Mol. Cell. Biol. 8:2896-2909, 1988, which isincorporated herein by reference) and the myosin light chainenhancer/promoter (Donoghue et al., Proc. Natl. Acad. Sci., USA88:5847-5851, 1991, which is incorporated herein by reference) are wellknown in the art. Other tissue specific promoters, as well as regulatoryelements only expressed during particular developmental stages of a cellor organism are well known in the art.

In additional embodiments, the regulatory elements contained in thenucleotide sequences used in or produced by the practice of theinvention can be one or more operators. A number of operators are knownin the art. An example of an operator suitable for use with theinvention is the tryptophan operator of the tryptophan operon of E.coli. The tryptophan repressor, when bound to two molecules oftryptophan, binds to the E. coli tryptophan operator and, when suitablypositioned with respect to the promoter, blocks transcription. Anotherexample of an operator suitable for use with the invention is operatorof the E. coli tetracycline operon. Components of the tetracyclineresistance system of E. coli have also been found to function ineukaryotic cells and have been used to regulate gene expression. Forexample, the tetracycline repressor, which binds to tetracyclineoperator in the absence of tetracycline and represses genetranscription, has been expressed in plant cells at sufficiently highconcentrations to repress transcription from a promoter containingtetracycline operator sequences (Gatz et al., Plants 2:397-404 (1992)).The tetracycline regulated expression systems are described, for examplein U.S. Pat. No. 5,789,156, the entire disclosure of which isincorporated herein by reference. Additional examples of operators whichcan be used with the invention include the Lac operator and the operatorof the molybdate transport operator/promoter system of E. coli (see,e.g., Cronin et al., Genes Dev. 15:1461-1467 (2001) and Grunden et al.,J. Biol. Chem., 274:24308-24315 (1999)).

Thus, in particular embodiments, the invention provides methods forpreparing nucleic acid molecules that contain one or more operatorswhich can be used to regulate expression in prokaryotic or eukaryoticcells. As one skilled in the art would recognize, when a nucleic acidmolecule which contains an operator is placed under conditions in whichtranscriptional machinery is present, either in vivo or in vitro,regulation of expression will often be modulated by contacting thenucleic acid molecule with a repressor and one or more metabolites whichfacilitate binding of an appropriate repressor to the operator. Thus,the invention further provides methods for preparing nucleic acidmolecules which encode repressors which modulate the function ofoperators, as well as nucleic acid molecules produced by these methods,compositions comprising these molecules, and uses of these molecules andcompositions.

Regulatory or other elements useful in generating a construct accordingto a method of the invention can be obtained in various ways. Inparticular, many of the elements are included in commercially availablevectors and can be isolated therefrom and can be modified to contain atopoisomerase recognition site at one or both ends, for example, using aPCR method as disclosed herein. In addition, the sequences of orencoding the elements useful herein generally are well known anddisclosed in publications. In many cases, the elements, for example,many transcriptional and translational regulatory elements, as well ascell compartmentalization domains, are relatively short sequences and,therefore, are amenable to chemical synthesis of the element or anucleotide sequence encoding the element. Thus, in one embodiment, anelement comprising a composition of the invention, useful in generatinga ds recombinant nucleic acid molecule according to a method of theinvention, or included within a kit of the invention, can be chemicallysynthesized and, if desired, can be synthesized to contain atopoisomerase recognition site at one or both ends of the element and,further, to contain an overhanging sequence following cleavage by a sitespecific topoisomerase.

A topoisomerase-charged vector can be generated in the following manner(Genome Res. 9: 383-392, 1999): A vector is linearized with arestriction enzyme that leaves “sticky ends”. Using a ligase such as T4DNA ligase, adapter oligonucleotides are ligated to both ends, and bothstrands, of the linearized DNA. The adapter oligonucleotides contain andposition a 5′-CCCTT-3′ Vacccinia topoisomerase type I recognitionsequence such that it can be cleaved by topoisomerase and trap thecovalent topoisomerase-DNA complex at each 3′ end of the vector. Theadapted vector is then incubated with purified Vaccinia topoisomeraseand an annealing oligonucleotide that complete the “topoisomerase sites”at each end of the vector. The annealing oligonucleotide acts to leave abreak, or nick, in the “bottom” strand opposite the last T in the5′-CCCTT-3′ containing oligonucleotide. The oligonucleotide adapterfragments that are “downstream” of the topoisomerase cleavage site (the“leaving groups”) are released upon topoisomerase cleavage and areremoved in the topoisomerase-vector purification process. In the absenceof the 5′ hydroxyl from the “leaving group”, topoisomerase is trapped ina covalent complex with the DNA ends to produce a topoisomerase-chargedvector.

Where nucleic acid molecules are to be covalently linked according to amethod of the invention, the nucleotide sequences generally areoperatively linked such that the recombinant nucleic acid molecule thatis generated has a desired structure and performs a desired function orencodes a desired expression product. As used herein, the term“operatively linked” means that two or more nucleotide sequences arepositioned with respect to each other such that they act as a unit toeffect a function attributable to one or both sequences or a combinationthereof. The term “operatively covalently linked” is used herein torefer to operatively linked nucleotide sequences generated according toa method of the invention for generating a ds recombinant nucleic acidmolecule covalently linked in one or both strands. For example, anucleotide sequence containing an open reading frame can be operativelylinked to a promoter such that the promoter confers its regulatoryeffect on the open reading frame similarly to the way in which it wouldeffect expression of an open reading frame that it normally isassociated with in a genome in a cell. Similarly, two or more nucleotidesequences comprising open reading frames can be operatively linked inframe such that, upon transcription and translation, a chimeric fusionpolypeptide is produced.

Although a ds recombinant nucleic acid molecule covalently linked in oneor both strands, generated according to a method of the inventiongenerally is linear, the construct generated also can be a circularizedds recombinant nucleic acid molecule. Furthermore, a circular dsrecombinant nucleic acid molecule can be generated such that it has thecharacteristics of a vector, and contains, for example, regulatoryelements required for replication in a prokaryotic host cell, aeukaryotic host cell, or both, and can contain a nucleotide sequenceencoding a polypeptide that confers antibiotic resistance or the like.An advantage of such a method is that the generated ds recombinantnucleic acid molecule, which is circularized according to a method ofthe invention, can be transformed or transfected into an appropriatehost cell, wherein the construct is amplified. Thus, in addition to anin vitro method such as PCR, which can be used to generate large amountsof a linear ds recombinant nucleic acid molecule generated according toa method of the invention, an in vivo method using a host cell can beused for obtaining a large amount of a circularized product generatedaccording to a method of the invention. Such elements includingbacterial origins of replication, antibiotic resistance genes, and thelike, which comprise a topoisomerase recognition site according to thepresent invention, can be useful components to include in a kit of theinvention as disclosed herein.

It should be recognized that a linear ds recombinant nucleic acidmolecule covalently linked in one or both strands, also can be clonedinto a vector, which can be a plasmid vector or a viral vector such as abacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vacciniavirus, semliki forest virus and adeno-associated virus vector, all ofwhich are well known and can be purchased from commercial sources(Promega, Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL,Gaithersburg Md.). If desired, the vector can be linearized and modifiedaccording to a method of the invention, for example, using a PCR method,to contain a topoisomerase recognition site, or cleavage productthereof, at one or both 3′ termini, or can be constructed by one skilledin the art (see, generally, Meth. Enzymol., Vol. 185, Goeddel, ed.(Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994;Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993; Kirshenbaum et al., J.Clin. Invest. 92:381-387, 1993; each of which is incorporated herein byreference).

Viral expression vectors can be particularly useful where a method ofthe invention is practiced for the purpose of generating a dsrecombinant nucleic acid molecule covalently linked in one or bothstrands, that is to be introduced into a cell, particularly a cell in asubject. Viral vectors provide the advantage that they can infect hostcells with relatively high efficiency and can infect specific cell typesor can be modified to infect particular cells in a host.

Viral vectors have been developed for use in particular host systems andinclude, for example, baculovirus vectors, which infect insect cells;retroviral vectors, other lentivirus vectors such as those based on thehuman immunodeficiency virus (HIV), adenovirus vectors, adeno-associatedvirus (AAV) vectors, herpesvirus vectors, vaccinia virus vectors, andthe like, which infect mammalian cells (see Miller and Rosman,BioTechniques 7:980-990, 1992; Anderson et al., Nature 392:25-30 Suppl.,1998; Verma and Somia, Nature 389:239-242, 1997; Wilson, New Engl. J.Med. 334:1185-1187 (1996), each of which is incorporated herein byreference). For example, a viral vector based on an HIV can be used toinfect T cells, a viral vector based on an adenovirus can be used, forexample, to infect respiratory epithelial cells, and a viral vectorbased on a herpesvirus can be used to infect neuronal cells. Othervectors, such as AAV vectors can have greater host cell range and,therefore, can be used to infect various cell types, although viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events.

A method of the invention can be used to operatively covalently link afirst nucleic acid molecule containing an open reading frame to a second(and other) nucleic acid molecule containing an open reading frame suchthat a nucleic acid molecule encoding a chimeric polypeptide isgenerated. The chimeric polypeptide comprises a fusion polypeptide, inwhich the two (or more) encoded peptides (or polypeptides) aretranslated into a single product, i.e., the peptides are covalentlylinked through a peptide bond. For example, a first nucleic acidmolecule can encode a cell compartmentalization domain, such as a plasmamembrane localization domain, a nuclear localization signal, amitochondrial membrane localization signal, an endoplasmic reticulumlocalization signal, or the like, or a protein transduction domain suchas the human immunodeficiency virus TAT protein transduction domain,which can facilitate translocation of a peptide linked thereto into acell (see Schwarze et al., Science 285:1569-1572, 1999; Derossi et al.,J. Biol. Chem. 271:18188, 1996; Hancock et al., EMBO J. 10:4033-4039,1991; Buss et al., Mol. Cell. Biol. 8:3960-3963, 1988; U.S. Pat. No.5,776,689 each of which is incorporated herein by reference). Such adomain can be useful to target a fusion polypeptide comprising thedomain and a polypeptide encoded by a second nucleic acid molecule, towhich it is covalently linked according to a method of the invention, toa particular compartment in the cell, or for secretion from or entryinto a cell. As such, the invention provides a means to generate dsrecombinant nucleic acid molecules covalently linked in both strandsthat encode a chimeric polypeptide.

A fusion polypeptide expressed from a nucleic acid molecule generatedaccording to a method of the invention also can comprise a peptidehaving the characteristic of a detectable label or a tag such that theexpress fusion polypeptide can be detected, isolated, or the like. Forexample, a nucleic acid molecule containing a topoisomerase recognitionsite, or cleavage product thereof, as disclosed herein, can encode anenzyme such as alkaline phosphatase, β-galactosidase, chloramphenicolacetyltransferase, luciferase, or other enzyme; or can encode a peptidetag such as a polyhistidine sequence (e.g., hexahistidine), a V5epitope, a c-myc epitope; a hemagglutinin A epitope, a FLAG epitope, orthe like. Expression of a fusion polypeptide comprising a detectablelabel can be detected using the appropriate reagent, for example, bydetecting light emission upon addition of luciferin to a fusionpolypeptide comprising luciferase, or by detecting binding of nickel ionto a fusion polypeptide comprising a polyhistidine tag. Similarly,isolation of a fusion polypeptide comprising a tag can be performed, forexample, by passing a fusion polypeptide comprising a myc epitope over acolumn having an anti-c-myc epitope antibody bound thereto, then elutingthe bound fusion polypeptide, or by passing a fusion polypeptidecomprising a polyhistidine tag over a nickel ion or cobalt ion affinitycolumn and eluting the bound fusion polypeptide. Methods for detectingor isolating such fusion polypeptides will be well known to those in theart, based on the selected detectable label or tag (see, for example,Hopp et al., BioTechnology 6:1204, 1988; U.S. Pat. No. 5,011,912; eachof which is incorporated herein by reference).

A method of the invention also can be used to detectably label anucleotide sequence with a chemical or small organic or inorganic moietysuch that the nucleotide sequence is useful as a probe. For example, anucleic acid molecule, which has a topoisomerase recognition site, orcleavage product thereof, at a 3′ terminus, can have bound thereto adetectable moiety such as a biotin, which can be detected using avidinor streptavidin, a fluorescent compound (e.g., Cy3, Cy5, Fam,fluorescein, or rhodamine), a radionuclide (e.g., sulfur-35,technicium-99, phosphorus-32, or tritium), a paramagnetic spin label(e.g., carbon-13), a chemiluminescent compound, or the like, such that,upon generating a covalently linked double stranded recombinant nucleicacid molecule according to a method of the invention, the generatednucleic acid molecule will be labeled. Methods of detectably labeling anucleotide sequence with such moieties are well known in the art (see,for example, Hermanson, “Bioconjugate Techniques” (Academic Press 1996),which is incorporated herein by reference). Furthermore, a detectablelabel can be used to allow capture of a ds nucleic acid molecule that isgenerated by the present invention. Finally, a detectable label, forexample biotin, can be used to block ligation of a topoisomerase-chargedend of a first nucleic acid molecule to a labeled end of a secondnucleic acid molecule, thus providing a method to direct ligation to theunlabelled end of the second nucleic acid molecule. It should berecognized that such elements as disclosed herein or otherwise known inthe art, including nucleotide sequences encoding cellcompartmentalization domains, or detectable labels or tags, orcomprising transcriptional or translation regulatory elements can beuseful components of a kit as disclosed herein.

A method of the invention provides a means to conveniently generate dsrecombinant nucleic acid molecules that encode chimeric polypeptidesuseful, for example, for performing a two hybrid assay. In such amethod, the first nucleic acid molecule encodes a polypeptide, or arelevant domain thereof, that is suspected of having or being examinedfor the ability to interact specifically with one or more otherpolypeptides. The first nucleic acid molecule is modified as disclosedherein to contain a topoisomerase recognition site at one or both endsand, if desired, a 5′ overhanging sequence. The second nucleic acidmolecule, to which the first nucleic acid molecule is to becovalently-linked according to a method of the invention, can encode atranscription activation domain or a DNA binding domain (Example 2.C),and contains a topoisomerase recognition site, or cleavage productthereof, and a 5′ overhanging sequence complementary to that at the endof the first nucleic acid molecule to which it is to be linked. Uponcontact with a topoisomerase, if the nucleotide sequences are notalready topoisomerase-charged, a first hybrid useful for performing atwo hybrid assay (see, for example, Fields and Song, Nature 340:245-246,1989; U.S. Pat. No. 5,283,173; Fearon et al., Proc. Natl. Acad. Sci.,USA 89:7958-7962, 1992; Chien et al., Proc. Natl. Acad. Sci., USA88:9578-9582, 1991; Young, Biol. Reprod. 58:302-311 (1998), each ofwhich is incorporated herein by reference), or modified form of a twohybrid assay such as the reverse two hybrid assay (Leanna and Hannink,Nucl. Acids Res. 24:3341-3347, 1996, which is incorporated herein byreference), the repressed transactivator system (U.S. Pat. No.5,885,779, which is incorporated herein by reference), the proteinrecruitment system (U.S. Pat. No. 5,776,689, which is incorporatedherein by reference), and the like, is generated. Similar methods areused to generate the second hybrid protein, which can comprise aplurality of polypeptides to be tested for the ability to interact withthe polypeptide, or domain thereof, of the first hybrid protein.

Similarly, such a method of generating a chimeric protein can beperformed according to a method of the current invention for generatinga ds recombinant nucleic acid molecule covalently linked in one strand,using first and second nucleic acid molecules comprising a site-specifictopoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site), or cleavage product thereof, at leastat one 5′ terminus of an end to be joined, wherein the nucleic acidmolecules can further comprise complementary 3′ overhangs upon cleavageby the topoisomerase.

Similarly, such a method of generating a chimeric protein can beperformed according to a method of the current invention for generatinga ds recombinant nucleic acid molecule covalently linked in both strandsusing first and second nucleic acid molecules comprising a topoisomeraserecognition site, or cleavage product thereof, at least at the 5′terminus of the ends to be joined, wherein the nucleic acid moleculescan further comprise complementary 3′ overhangs upon cleavage by thetopoisomerase; or one of the first or second nucleic acid molecules cancomprise topoisomerase recognition sites, or cleavage products thereof,at the 5′ terminus and the 3′ terminus of at least one end, and theother nucleic acid molecule can contain a 3′ hydroxyl group and a 5′hydroxyl group at the end to be joined, and wherein, upon cleavage bythe topoisomerases, the topoisomerase-charged nucleic acid molecule cancontain a 5′ or 3′ overhang that is complementary to, and facilitateshybridization to, a 5′ or 3′ overhang, respectively, or a blunt end, atthe end of the other nucleic acid molecule to be joined.

In an alternative embodiment, the present invention also provides amethod for the directional insertion of DNA fragments into cloning orexpression vectors with the ease and efficiency oftopoisomerase-mediated cloning. This invention also has advantages overcurrent cloning systems because it decreases the laborious screeningprocess necessary to identify cloned inserts in the desired orientation.This aspect of the invention consists, in its simplest form, of alinearized expression vector having a single topoisomerase moleculecovalently attached at both 3′ ends. At least one end of the linearizedvector contains a 5′ single-stranded overhang, while the opposite endcan be either blunt, possess a single 3′ T extension for T/A cloning, ormay itself contain a second 5′ single-stranded overhang sequence. Thesesingle-stranded sequence overhangs are alternatively referred to hereinas “SSS” and may consist of any convenient sequence.

Construction of a topoisomerase-charged cloning vector according to thisaspect of the invention may be accomplished, for example, byendonuclease digestion of the vector (which may be a pDONR vector (seeFIG. 32) or a pDEST vector (see FIG. 33)), followed by complementaryannealing of synthetic oligonucleotides and site-specific cleavage ofthe heteroduplex by Vaccinia topoisomerase I. Digestion of a vector withany compatible endonuclease creates specific sticky ends. Customoligonucleotides may be annealed to these sticky ends, and possesssequences that, following topoisomerase I modification, form custom endsof the vector (see FIGS. 32 and 33). The sequence and length of the SSSwill vary based on the desires of the user.

In one use of the TOPO SSS vectors provided by this aspect of thepresent invention, the DNA fragment to be inserted into the vector is aPCR product. Following PCR amplification with custom primers, theproduct can be directionally inserted into a topoisomerase I chargedcloning vector having a SSS on one or both ends of the insertion site.The custom primers may be designed such that at least one primer of agiven primer pair contains an additional sequence at its 5′ end. Theadded sequence may be designed to be complementary to the sequence ofthe single-stranded overhang in the vector. The complementarity betweenthe 5′ single-stranded overhang in the vector and the 5′ end of the PCRproduct mediates the directional insertion of the PCR product into thetopoisomerase-mediated vector. Specifically, since only one end of thevector and one end of the PCR product possess complimentary SSS regions,the insertion of the product is directional. Topoisomerase I catalyzesthe ligation of the PCR product to the vector.

This aspect of the invention also provides a modified cloning vector,having an overhanging single stranded piece of DNA, (the SSS) chargedwith topoisomerase, or “TOPO SSS vector”. The modified vector allows thedirectional insertion of PCR amplified, or otherwise suitable, openreading frames (ORF) for subsequent expression, and takes advantage ofthe efficiency of topoisomerase-mediated cloning.

As noted above, topoisomerases are a class of enzymes that modify thetopological state of DNA via the breakage and rejoining of DNA strands,(Shuman et al., U.S. Pat. No. 5,766,891, incorporated herein byreference). Vaccinia virus encodes a 314 aa type I topoisomerase enzymecapable of site-specific single-strand nicking of double stranded DNA,as well as 5′ hydroxyl driven religation. Site-specific type Itopoisomerases include, but are not limited to, viral topoisomerasessuch as pox virus topoisomerase. Examples of pox virus topoisomerasesinclude shope fibroma virus and ORF virus. Other site-specifictopoisomerases are well known to those skilled in the art and can beused to practice this invention.

Shuman teaches that Vaccinia topoisomerase binds to duplex DNA andcleaves the phosphodiester backbone of one strand while exhibiting ahigh level of sequence specificity. Cleavage occurs at a consensuspentapyrimidine element 5′-(C/T)CCTT-3′ or related sequences in thescissile strand. In one embodiment the scissile bond is situated in therange of 2-12 bp from the 3′ end of the duplex DNA. In anotherembodiment cleavable complex formation by Vaccinia topoisomeraserequires six duplex nucleotides upstream and two nucleotides downstreamof the cleavage site. Examples of Vaccinia topoisomerase cleavablesequences include, but are not limited to, +6/−6 duplex GCCCTTATTCCC(SEQ ID NO: 29), +8/−4 duplex TCGCCCTTATTC (SEQ ID NO: 30), +10/−2duplex TGTCGCCCTTAT (SEQ ID NO: 31), +11/−1 duplex GTGTCGCCCTTA (SEQ IDNO: 32).

Examples of other site-specific type I topoisomerases are well known inthe art. These enzymes are encoded by many organisms including, but notlimited to Saccharomyces cerevisiae, Saccharomyces pombe andTetrahymena, however these species' topoisomerase I enzymes have lessspecificity for a consensus sequence than does Vaccinia's. (Lynn, R. M.,Bjornsti, M., Caron, P. R. and Wang, J. C., (1989) Peptide sequencingand site-directed mutagenesis identify tyrosine-727 as the active sitetyrosine of Saccharomyces cerevisiae DNA topoisomerase I, Proc. Natl.Acad. Sci. USA, 86: 3559-3563), (Eng, W., Pandit, S. D., and Sternglanz,R., (1989) Mapping of the active site tyrosine of eukaryotic DNAtopoisomerase I, J. Biol. Chem., 264: 13373-13376) and (Busk, H.,Thomsen, B., Bonven, B. J., Nielsen, O. F., and Westergaard, O. (1987)Preferential relaxation of supercoiled DNA containing a hexadecamericrecognition for topoisomerase I, Nature, 327: 638-640), respectively.

As used herein with regard to this aspect of the invention, the termdonor signifies a duplex DNA which contains a 5′-CCCTT cleavage sitenear the 3′ end, and the term acceptor signifies a duplex DNA whichcontains a 5′-OH terminus. Once covalently activated by topoisomerasethe donor will be transferred to those acceptors to which it has SSScomplementation.

According to this aspect of the present invention,topoisomerase-modified vectors are further adapted to contain at leastone 5′ single-stranded overhang sequence to facilitate the directionalinsertion of DNA segments. In a preferred embodiment, the segment to becloned is a PCR product constituting an open reading frame (ORF) whichwill be expressed from the resultant recombinant vector. The primersused for amplifying the ORF are designed such that at least one primerof the primer pair contains an additional sequence at its 5′ end. Thissequence is designed to be complementary to the sequence of the 5′single-stranded overhang present in the topoisomerase-modified vector ofthe present invention.

Certain preferred, but non-exclusive, embodiments according to thisaspect of the present invention are described in detail below inExamples 5-8.

Nucleic acid molecules assembled using methods of the invention eithermay be used directly or may be amplified and then used for any number ofpurposes. With reference to FIG. 34, nucleic acid segments to beassembled using methods of the invention may be generated by any numberof methods. For example, these segments may be obtained by any methodknown in the art. In instances where the nucleic acid segments do nothave one or more (e.g., one, two, three, four, etc.) termini and/orregions suitable for assembly using methods of the invention, suchtermini and/or regions may be added. Suitable termini and/or regions maybe added, for example, by amplifying nucleic acids using PCR or by theaddition of one or more (e.g., one, two, three, four, etc.) adapterlinkers (e.g., adapter linkers which contain one or more topoisomeraserecognition sites). Nucleic acid segments having suitable termini and/orregions may then be assembled using methods of the invention describedelsewhere herein.

As shown in FIG. 34, once assembled, the linked nucleic acid segmentsmay be amplified (e.g., in vivo or in vitro) and then used in any numberof methods or processes, many of which are described elsewhere herein.Alternatively, the assembled nucleic acid segments may be used directlyfor applications such as in vitro transcription/translation,recombinational cloning, or for transforming or transfecting cells. Theinvention thus provides versatile compositions and methods formanipulating nucleic acids.

As also indicated in FIG. 34, the invention further provides methods forlinking nucleic acid segments which then may be used in any number ofmethods or processes. As one example of such a method, the initialligation products generated by the first step set out in Figure A, whichis referred to here solely for illustrative purposes, are directlytranscribed (e.g., used for in vitro transcription). This process isfacilitated by the fact that the nucleic acid which is transcribed iscovalently linked in both strands at the junction point between thelinker containing the T7 promoter element and the PCR product. Further,transcription of the linkage products from the T7 promoter results inthe production of both sense and antisense RNA which can be used toform, for example, double stranded RNA. This double stranded RNA can beused for inhibiting gene expression. In particular, methods of theinvention may be used to produce double stranded RNA for RNAiapplications. Such RNAi molecules may be prepared from RNA moleculesprepared in two separate tubes and then mixed or in the same tube. Inthe first case, transcription of sense strand and antisense strand RNAmay occur after DNA molecules which encode these strand have beenseparated and placed in separate tubes. In the second case,transcription of both sense strand and antisense strand RNA may occur inthe same tube. Thus, the invention also provides one and two tubemethods for the preparation RNA for, for example, the preparation ofRNAi.

As one skilled in the art would recognize, any number of variations ofthe above are possible and within the scope of the invention. Forexample, a promoter other than a T7 promoter may be used. Further, anyof the nucleic acid molecules described above, as well as elsewhereherein, may be designed to contain one or more recombination sites whichcan then be used to connect these molecules with other nucleic acidmolecules (e.g., other nucleic acid molecules with cognate recombinationsites).

The invention provides compositions and methods for linking nucleic acidmolecules using topoisomerase and recombination. In particularembodiments of the invention, nucleic acid molecules undergo one or more(e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.)recombination reactions and are then linked to one or more (e.g., one,two, three, four, five, six, seven, eight, nine, ten, etc.) othernucleic acid molecules by methods involving covalent linking of strandscatalyzed by one or more (e.g. one, two, three, four, etc.)topoisomerases. In other embodiments, nucleic acid molecules are linkedto other nucleic acid molecules by methods involving covalent linking ofstrands catalyzed by one or more (e.g., one, two, three, four, etc.)topoisomerases and then undergo one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, etc.) recombination reactions.As one skilled in the art would recognize, the invention is not tied toany particular order of topoisomerase-mediated linkage of nucleic acidmolecules or recombination reactions. Thus, in general, the invention isdirected to compositions and methods for performing both recombinationreactions and linking nucleic acid segments using topoisomerases.

The invention thus also provides adapter-linker molecules for use inaccordance with the methods and compositions of the invention. Theadapter linkers that are provided by, and that may be used in connectionwith, the present invention can contain both a topoisomerase site and arecombination site. One example of a process of the invention is set outschematically in FIG. 35. FIG. 35 shows a process which involves theconnection of a topoisomerase-adapted nucleic acid segment (“adapterlinker”) which contains a single recombination site to another nucleicacid segment, referred to as an insert. These two nucleic acid segmentsmay be connected by any topoisomerase-mediated process described herein.

Adapter linkers of the invention may comprise (1) one or morerecombination sites and/or (2) one or more topoisomerase recognitionsites or one or more topoisomerases. In particular embodiments, at leastone of the one or more recombination sites of the adapter linkers willbe located within zero, one, two, three, four, five, six, seven, eight,nine, ten, fifteen, or twenty nucleotides of at least one of the one ormore topoisomerase recognition site or one or more topoisomerase. Inspecific embodiments, recombination sites present in adapter linkers ofthe invention are attL, attB, attP, or attL recombination sites. Inadditional specific embodiments, the topoisomerase recognition sitesrecognition are recognition sites for type IB topoisomerases, type IAtopoisomerases or type II topoisomerases, or the topoisomerases are typeIB topoisomerases, type IA topoisomerases or type II topoisomerases. Inaddition, topoisomerase recognition sites or topoisomerases may belocated, with respect to recombination sites, in adapter linkers of theinvention such that upon recombination, particular recombination sitesbecome associated with the product molecules. For example, atopoisomerase recognition site may be located on either end of an attLsite in an adapter linker such that when the linker is attached to anucleic acid molecule and recombination occurs, either an attB or anattP site is generated on the nucleic acid molecule to which the adapterlinker was attached. Thus, adapter linkers may contain toposiomeraserecognition sites and/or topoisomerases positioned, with respect torecombination sites, such that upon ligation to a nucleic acid moleculeand recombination any number of variations of recombination sites arepresent on the product nucleic acid molecules. Examples of suchrecombination sites include attL, attB, attP, and attR recombinationsites.

The invention further provides methods for linking any number of nucleicacid segments using adapter linkers which contain recombination siteshaving the same or different specificities, as well as adapter linkerswhich contain recombination sites having the same or differentspecificities and kits which contain such adapter linkers. For example,three separate PCR products, referred to as segments A, B, and C, may belinked to adapter linkers such that attL1 and attL3 sites are present atthe ends of segment A, attR3 and attR4 sites are present at the ends ofsegment B, and attL4 and attL2 sites are present at the ends of segmentC. Thus, upon recombination with a linearized vector which containsattR1 and attR2 recombination sites at or near the termini, all threePCR products are joined to each other and inserted into the vector togenerate a circularized nucleic acid molecule. Any number of variationsof the above are possible and are within the scope of the invention.

The invention further includes sets of two or more (e.g., two, three,four, five, six, seven eight, nine, etc.) adapter linkers which contain(1) one or more recombination sites having the same or differentspecificities and/or (2) one or more topoisomerases or toposiomeraserecognition sites, as well as methods for using these sets of adapterlinkers to generate nucleic acid molecules which contain one or morerecombination sites, compositions comprising such adapter linker sets orindividual member of these sets, nucleic acid molecules which have beenadapted with one or more adapter linkers of these sets, and methods forusing these nucleic acid molecules.

After topoisomerase-mediated assembly, the assembled nucleic acidmolecule may be recombined with another nucleic acid segment whichcontains one or more (e.g., one, two, three, four, etc.) suitablerecombination sites. The recombination sites shown in FIG. 35 are attL1and attR1 sites but any suitable recombination sites may be used (e.g.lox sites, attR sites, attL sites, attB sites, attP sites, etc.).Additional suitable recombination sites are described elsewhere herein.

The invention thus includes methods for generating nucleic acidmolecules using topoisomerase recognition sites and recombination siteswith recombine with each other. The invention also includes nucleic acidmolecules prepared by and used in methods of the invention, as well asmethods for using nucleic acid molecules generated by methods describedherein.

The invention further includes methods for generating nucleic acidmolecules using multiple (e.g., two, three, four, five, six, seven,eight, nine, ten, etc.) recombination sites and topoisomeraserecognition sites, as well as nucleic acid molecules prepared by andused in such methods. Further, these recombination sites may havemultiple (e.g., two, three, four, five, six, seven, eight, nine, ten,etc.) specificities. In addition, the topoisomerase recognition sitesmay be designed to generate termini which will result in the connectionof these termini to different nucleic acid segments. For example, thesetermini may be designed to generate different “sticky ends” uponcleavage with a topoisomerase.

Another example of methods described above is shown in FIG. 36. FIG. 36shows a process in which two nucleic acid segments are connected using aprocess which involves topoisomerase-mediated covalent linkage ofstrands of the termini of the nucleic acid segments. The resultingnucleic acid molecule then undergoes recombination, which results in (1)the topoisomerase assembled nucleic acid molecule becoming linked to anucleic acid segment which contains an origin of replication and (2)replacement of a negative selection marker (e.g., a ccdB gene) with apromoter. The recombined nucleic acid product is then connected to anucleic acid segment which is topoisomerase adapted at both termini andcontains a positive selection marker. This last step results in thenucleic acid molecule being circularized.

The circularized nucleic acid end product shown in FIG. 36 may beintroduced into host cells, which may be prokaryotic (e.g., bacterial)or eukaryotic (e.g., yeast, plant, animal (including mammalian, such ashuman)) cells such as those described elsewhere herein. Further, cellswhich contain this end product can be selected for using positive andnegative selection. Thus, for example, cells which have acquired anucleic acid molecule wherein the negative selection marker has not beenreplaced by the promoter will be selected against. The invention furtherincludes methods and compositions similar to those set out in FIGS. 35and 36 in which any number of the steps and components are varied.Examples of steps and components which may be varied are describedelsewhere herein. The invention further includes methods for usingnucleic acid molecules generated by methods described above.

As one skilled in the art would recognize, nucleic acid segments used inprocesses such as those shown in FIGS. 35 and 36 could contain anynumber of different elements. For example, a positive selection markercould be substituted for the promoters shown in FIG. 36. Further, theinsert shown in FIG. 35 may contain nucleic acid which has any number offunctionalities. In particular, when the insert contains a regions whichis transcribed, the transcript can be a mRNA or an RNA which serves afunction in the absence of translation. Examples of RNA which serves afunction in the absence of translation include transfer RNAs (e.g.,suppressor tRNAs), antisense RNAs, ribosomal RNAs, and ribozymes.Additionally, more than one of the nucleic acid segments connectedand/or recombined by methods of the invention may contain all or part ofone or more (e.g., one, two, three, four, five, six, seven, etc.) openreading frames. In such instances, nucleic acid segments may beconnected to each other such that transcription and translation resultin the production of one or more fusion proteins. Additional nucleicacid elements which can be used in methods of the invention aredescribed elsewhere herein.

Once a nucleic acid molecule, such as the end product of the processshown in FIG. 35, has been generated by methods of the invention, thenucleic acid molecule may optionally be connected to one or more (e.g.,one, two, three, four, etc.) other nucleic acid molecules or may becircularized by joining of the termini to each other. Further, whenthree or more nucleic acid molecules are connected to each other bymethods of the invention, the termini of various intermediate moleculesor the end product may be joined to each other to circularize thesemolecules.

The invention further provides compositions and methods for performinghomologous recombination and for producing transgenic animals. Genetargeting by homologous recombination between an exogenous DNA constructand cognate chromosomal sequences allows precise modifications to bemade at predetermined sites in the genome. Gene targeting iswell-established in, e.g., mouse embryonic stem (ES) cells, and has beenused to effect modifications in a large number of murine genes. (Seee.g., Brandon et al., Curr. Biol. 5:625-634, 758-765, 873-881 (1995)).Gene targeting can also be accomplished in somatic cells. (See e.g.,Itzhaki et al., Nat. Genet. 15:258-265 (1997)). Cells that have beenmodified by gene targeting via homologous recombination can then bemanipulated by methods known in the art to establish transgenic animals.

One example of a composition of the invention that can be used inhomologous recombination applications is the end product nucleic acidmolecule set out in FIG. 37. FIG. 37 further shows an example of amethod for preparing such compositions. In particular, FIG. 37 shows thelinkage of topoisomerase adapted nucleic acid segments to anon-topoisomerase adapted nucleic acid segment. In this instance, thenucleic acid segment which the designer of the nucleic acid end productseeks to integrate into a chromosome, referred to here as an insert, isflanked by regions which contain (1) a positive selection marker and (2)a negative selection marker positioned between two recombination sites.Recombination may then be used to replace the two negative selectionmarkers with nucleic acid having homology to a chromosomal region intowhich the end product is to integrate (labeled “HR1” and “HR2” in FIG.37).

Regions of homology used in the practice of the invention will vary withthe chromosomes of cells into which nucleic acid molecules are tointegrate. Further, in many instances, regions of homology will beselected to facilitate integration into cells of a particular organism.Such an organism may be unicellular organism (e.g., a yeast, aprotozoan, etc.) or multicellular organism (e.g., a plant, an animal,etc.).

The invention thus provides nucleic acid molecules and compositions forperforming homologous recombination and cells produced via homologousrecombination involving these molecules and compositions. Methods of thepresent invention can be used in the linking of multiple nucleic acidsegments. FIG. 38, for example, shows a schematic representation of thelinking of four nucleic acid segments using toposiomerase to generate alinear nucleic acid molecule with recombination sites (labeled “L1” and“L2”) located near the termini. In the first step, topoisomerase adaptednucleic acid segment which contains an attL1 recombination site and anattL2 recombination site are linked to two other nucleic acid segmentsusing topoisomerase. In this particular instance, each strand of thetermini which are joined to each other is covalently linked to atopoisomerase molecule. Thus, upon toposiomerase mediated linkage of thenucleic acid strands, no nicks are present at the junction points. Inthe second step, the topoisomerase assembled nucleic acid segments arecontacted with another nucleic acid segment which contains an origin ofreplication (labeled “ori”), a positive selection marker (labeled “PM”),an attR1 recombination site, and an attR2 recombination site in thepresence of LR CLONASE™ under conditions which allow for recombinationbetween the attL and attR recombination sites. In certain such methods,for example, TOPO-adapted vectors are incubated with one or more nucleicacid segments (e.g., one or more PCR products) at room temperature(e.g., about 20-20° C.) for about 5-30 (and preferably about 10)minutes; the reaction is then heat-treated by incubation at about 80° C.for about 20 minutes, and the reaction mixture then used in a standardLR reaction according to manufacturer's instructions (InvitrogenCorporation, Carlsbad, Calif.), except the incubation time for the LRreaction is increased to about 3 hours. Recombination results in theformation of a circular nucleic acid molecule which contains the variousstarting nucleic acid segments separated from the origin and selectionmarker by attB1 and attB2 recombination sites. As one skilled in the artwould recognize, any suitable recombination sites could be used in placeof the aut recombination sites shown in this figure. The invention thusalso provides compositions comprising such nucleic acids, compositionsused for producing such nucleic acids, and uses of such nucleic acidsand compositions in the recombination and topoisomerase-mediated joiningmethods of the invention described elsewhere herein.

The invention further provides nucleic acid molecules suitable forperforming cloning reactions in which a first nucleic acid molecule,which shares one or more region of homology with a second nucleic acidmolecule, is used to insert nucleic acid from the second nucleic acidmolecule into the first nucleic acid molecule. The invention furtherprovides compositions and methods for performing such cloning reactions.

One example of a process referred to above is RecE/T cloning, which isdescribed in PCT Publication WO 01/04288, the entire disclosure of whichis incorporated herein by reference. Typically, in RecE/T cloning, alinear first nucleic acid molecule (e.g., a vector) is introduced into acell which contains (1) regions at the termini that share homology withtwo separate, nearby regions (e.g., nucleic acid regions which are about20 to about 30, about 20 to about 40, about 20 to about 50, about 30 toabout 40, about 40 to about 50, about 40 to about 60, about 40 to about80, about 50 to about 90, etc. nucleotides in length) of a nucleic acidmolecule present in the cell (e.g., a plasmid, a bacterial artificialchromosome, a natural chromosome, etc.), referred to here as “a secondnucleic acid molecule”, (2) a selection marker, and (3) an origin ofreplication. The linear first nucleic acid molecule will generally onlyreplicate if it becomes circularized. Further, the first nucleic acidmolecule will typically become circularized when it has undergonerecombination with the second nucleic acid molecule and acquired nucleicacid from the second nucleic acid molecule which is intervening betweenthe regions of homology. In such embodiments, the regions of homology inthe first nucleic acid molecule will typically be in a reverseorientation as compared to the second nucleic acid molecule. Generally,the cell in which recombination occurs will be one which expresses arecombinase such as RecE/T or RecAlpha/Beta. Thus, the inventionprovides, in part, methods for performing RecE/T cloning, nucleic acidmolecules prepared by such methods, compositions comprising such nucleicacid molecules, and methods for using such nucleic acid molecules andcompositions.

Modifications of the RecE/T process may be employed to generate a numberof different end products. For example, when the regions of homology arearranged in various ways, the first nucleic acid molecule can bedesigned to (1) insert into the second nucleic acid molecule, or (2)delete nucleic acid from the second nucleic acid molecule. Typically,when insertion of the second nucleic acid molecule into the secondnucleic acid molecule is desired, the regions of homology of the firstnucleic acid molecule will be in the same orientation with respect tothe regions of homology in the second nucleic acid molecule. Further,when deletion of nucleic acid from the second nucleic acid molecule isdesired, the regions of homology of the first nucleic acid molecule willgenerally be in an inverse orientation with respect to the regions ofhomology in the second nucleic acid molecule. Also, when insertion ofthe first nucleic acid molecule into the second nucleic acid molecule isdesired, typically the first nucleic acid molecule will not contain anorigin of replication. The invention provides methods for performing theabove processes. The invention also provides nucleic acid molecules andcompositions for use in the above processes.

The present invention can also be used to link two nucleic acid segmentsin a single step process using topoisomerase and recombination sites togenerate a circular nucleic acid molecule. An example of this embodimentis depicted in FIG. 39 where one of the nucleic acid segments containsan attL1 recombination site (labeled “L1”), a promoter (labeled “P”),and toposiomerase molecule covalently linked to one terminus. The othernucleic acid segment contains an attR1 recombination site (labeled“R1”), an open reading frame (labeled “ORF”), an origin of replication(labeled “ORI”), a positive selection marker (labeled “PM”), andtopoisomerase molecule covalently linked to one terminus. Thus, whenthese two nucleic acid segments are contacted with each other in thepresence of LR CLONASE™ under conditions which allow for recombinationbetween the attL and attR recombination sites and topoisomerase mediatedlinkage of nucleic acid strands, a circular molecule is formed havingthe structure indicated. In certain such methods, for example,TOPO-adapted vectors are incubated with one or more nucleic acidsegments (e.g., one or more PCR products) at room temperature (e.g.,about 20-20° C.) for about 5-30 (and preferably about 10) minutes; thereaction is then heat-treated by incubation at about 80° C. for about 20minutes, and the reaction mixture then used in a standard LR reactionaccording to manufacturer's instructions (Invitrogen Corporation,Carlsbad, Calif.), except the incubation time for the LR reaction isincreased to about 3 hours. As one skilled in the art would recognize,any suitable recombination sites could be used in place of the attrecombination sites shown in this figure.

The present invention can also be used to link two nucleic acid segmentsusing toposiomerase mediated methods to generate a circular nucleic acidmolecule. A schematic representation of one embodiment of this aspect ofthe invention is illustrated in FIG. 40. As shown in FIG. 40, thecircular molecule contains an open reading frame (labeled “ORF”)positioned between attL1 and attL2 recombination site (labeled “L1” and“L2”). The topoisomerase assembled product then undergoes recombinationwith another circular molecule which contains attR1 and attR2recombination sites to generate a third circular nucleic acid moleculewhich contains the open reading frame positioned between attB1 and attB2recombination sites. Further, the open reading frame is operably linkedto a promoter. Thus, the final nucleic acid molecule produced by thisprocess is an expression construct. As one skilled in the art wouldrecognize, any suitable recombination sites could be used in place ofthe att recombination sites shown in this figure.

As disclosed herein, a first nucleic acid molecule can be one of aplurality of nucleotide sequences, for example, a cDNA library, acombinatorial library of nucleotide sequences, or a population ofvariegated nucleotide sequences. As such, a particularly usefulembodiment of a method of the invention is in generating recombinantpolynucleotides encoding chimeric polypeptides for performing a highthroughput two hybrid assay for identifying protein-protein interactionsthat occur among populations of polypeptides (see U.S. Pat. No.6,057,101 and U.S. Pat. No. 6,083,693, each of which is incorporatedherein by reference). In such a method, two populations (pluralities) ofnucleotide sequences encoding polypeptides are examined, each pluralityhaving a complexity of from a few related but different nucleotidesequences to as high as tens of thousands of such sequences. Byperforming a method of the invention, for example, using a PCR primerpair to amplify each nucleotide sequence in the plurality, wherein atleast one primer of the PCR primer pair comprises (a) at least onetopoisomerase recognition site or complement thereof or (b) at least onerecombination site, covalently linked recombinant polynucleotidesencoding a population of chimeric bait polypeptides and a population ofchimeric prey polypeptides readily can be generated by contacting theamplified pluralities of nucleotide sequences, each of which comprises(a) at least one topoisomerase recognition site, with at least onetopoisomerase and a nucleotide sequence, which contains at least onetopoisomerase recognition site and encodes a transcription activationdomain or a DNA binding domain or (b) at least one recombination site,with at least one topoisomerase and a nucleotide sequence, whichcontains at least one recombination site and encodes a transcriptionactivation domain or a DNA binding domain.

In practicing a method of the invention, a first nucleic acid moleculealso can encode a ribonucleic acid (RNA) molecule, which can function,for example, as a riboprobe, an antisense nucleotide sequence, aribozyme, or a triplexing nucleotide sequence, or can be used in an invitro translation reaction, and the second nucleic acid molecule canencode a regulatory element useful for expressing an RNA from the firstnucleotide sequence (see Example 2.A). For example, where it is desiredto produce a large amount of RNA, a second nucleic acid moleculecomponent for performing a method of the invention can comprise an RNApolymerase promoter such as a T7, T3 or SP6 RNA polymerase promoter.Where the RNA molecule is to be expressed in a cell, for example, anantisense molecule to be expressed in a mammalian cell, the second (orother) nucleic acid molecule can include a promoter that is active in amammalian cell, particularly a tissue specific promoter, which is activeonly in a target cell. Furthermore, where the RNA molecule is to betranslated, for example, in a coupled in vitro transcription/translationreaction, the first nucleotide sequence or second (or other) nucleotidesequence can contain appropriate translational regulatory elements (seeExample 2.B).

Methods of the invention may also be used to produce constructs whichallow for silencing of genes in vivo. One method of silencing genesinvolves the production of double-stranded RNA, termed RNA interference(RNAi). (See, e.g., Mette et al., EMBO J, 19:5194-5201 (2000)). Themechanism by which RNAi is believed to function, which is reviewed inFjose et al., Biotechnol. Annu. Rev. 7:31-57 (2001), appears to be basedon the ability of double stranded RNA to induce the degradation ofspecific RNA molecules. This mechanism is reported to involve theconversion of double-stranded RNA into short RNAs that directribonucleases to homologous RNA targets (e.g., mRNA targets). Methods ofthe invention can be used in a number of ways to produce molecules suchas RNAi. Thus, expression products of nucleic acid molecules of theinvention can be used to silence gene expression.

One example of a nucleic acid molecule designed to produce RNAi is amolecule in which a nucleic acid segment is linked to one or morepromoters such that RNA corresponding to both strands are produced astwo separate transcripts or as part of the same transcript. For example,a nucleic acid molecule could be prepared using methods of the inventionwherein two copies of an open reading frame are connected by anintervening nucleic acid segment with two promoters that drivetranscription in different directions. Thus, one of the promoters drivestranscription of sense strand mRNA and the other promoter drivestranscription of antisense mRNA. Another example of a nucleic acidmolecule which could be used to produce RNAi is one in which an openreading frame is flanked on each end by promoters which, drivetranscription of the open reading frame in opposing directions. As athird example, doubles stranded RNA can be produced from a nucleic acidmolecule which encode RNA having a “snapback” region (e.g., a regionthat is six, seven, eight, nine ten, etc. nucleotides in length) at oneterminus. Thus, an RNA transcript of this type will form a hairpin turnat or near one terminus. When such an RNA molecule is incubated, underappropriate conditions, in the presence of an RNA dependent RNApolymerase, the double stranded region formed by the hairpin can be usedto prime second strand synthesis to form double stranded RNA molecule.

Nucleic acid segments designed to produce RNAi, such as the nucleic acidmolecules described above, need not correspond to the full-length geneor open reading frame. For example, when the nucleic acid segmentcorresponds to all or part of an ORF or encode an RNA molecule whichdoes not correspond to all or part of an ORF, the segment may onlycorrespond to part of the ORF (e.g., about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 40, about50, about 60, etc. nucleotides at the 5′ or 3′ end of the ORF).

Thus, in particular embodiments, the invention provides methods forpreparing nucleic acid molecules comprising at least three segments. Insome embodiments, at least two of these segments share at least oneregion of sequence identity (e.g., a region at least about 15, at leastabout 16, at least about 17, at least about 18, at least about 19, atleast about 20, at least about 21, at least about 22, at least about 23,at least about 24, at least about 25, at least about 26, at least about27, at least about 28, at least about 29, at least about 30, at leastabout 40, at least about 50, at least about 60, at least about 70, atleast about 80, at least about 90, at least about 100 nucleotides, etc.nucleotides in length). In other embodiments, one nucleic acid segmentis flanked by a region which can confer transcription of the interiorportion of the molecule in opposing directions (e.g., to produce senseand antisense transcripts). The invention further provides nucleic acidmolecules prepared by methods of the invention and the use of suchmolecules to either inhibit gene expression or facilitate thedegradation of specific RNA molecules.

The invention further includes methods for preparing nucleic acidmolecules which express one or more RNA molecules which can be used toprepare double stranded RNA having overhangs on one or both ends. Forexample, methods of the invention can be used to express two singlestranded RNA molecules which are 21 nucleotides in length and sharesequence complementarity over 19 of their nucleotides. Thus, when thesetwo single stranded RNA molecules hybridize to each other, there will bea two nucleotide overhang on each end. Double stranded RNA moleculessimilar to those described above have been shown to be capable ofinhibiting gene expression when introduced into mammalian cells(Elbashir et al., Nature 411:494-498 (2001)).

The invention thus includes methods for generating nucleic acidmolecules which can be used to produce short RNA molecules, as well asRNA molecules produced by nucleic acid molecules prepared by thesemethods and methods for preparing these RNA molecules. These short RNAmolecules will typically be about 15, about 16, about 17, about 18,about 19, about 20, about 21, about 22, about 23, about 24, about 25,about 26, about 27, about 28, about 29, about 30 nucleotides in length.Further, these short RNA molecules will typically be between from about15 to about 30, from about 15 to about 25, from about 15 to about 24,from about 23 to about 22, from about 15 to about 21, from about 15 toabout 20, from about 15 to about 19, from about 15 to about 18, fromabout 20 to about 30, from about 20 to about 28, from about 20 to about25, from about 20 to about 24, from about 20 to about 23, from about 20to about 22, or from about 20 to about 21 nucleotides in length.

The invention further includes methods for generating nucleic acidmolecules which can be used to produce short double stranded RNAmolecules, as well as RNA molecules produced by nucleic acid moleculesprepared by these methods. These short double stranded RNA molecules maycomprise a double stranded region which is about 10, about 12, about 14,about 15, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30 nucleotides in length. Further, the double strandedregion of these RNA molecules may be between from about 10 to about 30,from about 10 to about 25, from about 10 to about 20, from about 10 toabout 18, from about 10 to about 17, from about 15 to about 30, fromabout 15 to about 25, from about 15 to about 24, from about 23 to about22, from about 15 to about 21, from about 15 to about 20, from about 15to about 19, from about 15 to about 18, from about 20 to about 30, fromabout 20 to about 28, from about 20 to about 25, from about 20 to about24, from about 20 to about 23, from about 20 to about 22, or from about20 to about 21 nucleotides in length. Further, these double stranded RNAmolecules may comprise overhangs at one or both termini which are about1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8nucleotides in length and/or are between from about 1 to about 10, fromabout 1 to about 8, from about 1 to about 6, from about 1 to about 4,from about 1 to about 2, from about 2 to about 10, from about 2 to about8, from about 2 to about 6, or from about 2 to about 4 nucleotides inlength.

The invention also provides methods for preparing nucleic acid moleculeswhich can be used to express antisense RNA (e.g., antisense mRNA).Methods similar to those described above for the production of nucleicacid molecules which can be used for RNAi may be employed; however, onlythe antisense strand will typically be transcribed in molecules preparedby methods of the invention which may be used to generate antisense RNA.

In related embodiments, promoters which drive transcription of the senseRNA or antisense RNA can be either constitutive (e.g., CMV promoter,SV40 promoter, etc.), inducible (e.g., a metallothionein promoter,etc.), or repressible. Thus, for example, two different induciblepromoters can be used to drive transcription of sense RNA and antisenseRNA. In such an instance, promoter activation can be used to induceproduction of sense RNA, antisense RNA, or both sense RNA and antisenseRNA. Further, the amount of sense RNA and/or antisense RNA produced canbe related by using, for example, graduated induction and/orderepression of the promoters.

Gene silencing methods involving the use of compounds such as RNAi andantisense RNA, for examples, are particularly useful for identifyinggene functions. More specifically, gene silencing methods can be used toreduce or prevent the expression of one or more genes in a cell ororganism. Phenotypic manifestations associated with the selectiveinhibition of gene functions can then be used to assign role to the“silenced” gene or genes. As an example, Chuang et al., Proc. Natl.Acad. Sci. (USA) 97:4985-4990 (2000), have demonstrated that in vivoproduction of RNAi can alter gene activity in Arabidopsis thaliana.Thus, the invention provides methods for regulating expression ofnucleic acid molecules in cells and tissues comprising the expression ofRNAi and antisense RNA. The invention further provides methods forpreparing nucleic acid molecules which can be used to produce RNAcorresponding to one or both strands of a DNA molecule.

The invention thus provides methods for regulating expression of nucleicacid molecules in vivo (e.g., in cells and tissues) and/or in vitrocomprising the expression of sense RNA and/or antisense RNA. Theinvention further provides methods for preparing nucleic acid moleculeswhich can be used to produce RNA corresponding to one or both strands ofa nucleic acid molecule (e.g., a DNA molecule). The invention alsoprovides compositions for performing the methods described above andnucleic acid molecules produced by the above methods (e.g., RNA and DNAmolecules).

The invention also relates to compounds and methods for gene silencinginvolving ribozymes. In particular, the invention provides antisenseRNA/ribozymes fusions, which comprise 1) antisense RNA corresponding toa target gene and 2) one or more ribozymes that cleave RNA (e.g.,hammerhead ribozyme, hairpin ribozyme, delta ribozyme, TetrahymenaL-21-ribozyme, etc.). Further provided by the invention are vectors thatexpress such fusions, methods for producing such vectors, and methodsfor using such vector to suppress gene expression.

Expression of antisense molecules fused to ribozymes can be used, forexample, to cleave specific RNA molecules in a cell because theantisense RNA portion of the transcript can be designed to hybridize toparticular “mRNA molecules. Further, the ribozyme portion of thetranscript can be designed to cleave the RNA molecule to which it hashybridized. For example, the ribozyme can be one which cleaves doublestranded RNA (e.g., a Tetrahymena L-21 ribozyme).

A method of the invention can be particularly useful for generating anexpressible ds recombinant nucleic acid molecule that can be inserted ina site specific manner into a target DNA sequence. The target DNAsequence can be any DNA sequence, particularly a genomic DNA sequence,and preferably a gene for which some or all of the nucleotide sequenceis known. The method can be performed utilizing a first nucleic acidmolecule, which has a first end and a second end and encodes apolypeptide, for example, a selectable marker, wherein the first nucleicacid molecule comprises at least one topoisomerase recognition siteand/or at least one recombination site or cleavage product thereof atthe 3′ terminus of each end and, optionally, a hydroxyl group at the 5′terminus of each end, and wherein, preferably, the 5′ termini comprise5′ overhanging sequences, which are different from each other; andcovalently linking the first nucleic acid molecule to first and secondPCR amplification products according to a method of the invention. Thefirst and second amplification products are generated from sequencesupstream and downstream of the site at which the construct is to beinserted, and each amplification product contains at least onetopoisomerase recognition site and optionally at least one recombinationsite, preferably, a 5′ overhanging sequence, which is generatedfollowing contact with the site specific topoisomerase. Preferably, thefirst and second amplification products have different 5′ overhangingsequences such that each can be linked to a predetermined end of thefirst nucleic acid molecule. Such a method similarly can be performedusing a ds amplification product comprising at least one topoisomeraserecognition site and, optionally, at least one recombination site, orcleavage product thereof, at the 5′ terminus of one or both ends,wherein, upon cleavage by the topoisomerase, the topoisomerase-chargedmolecule can comprise a 3′ overhang at one or both ends containing thetopoisomerase. In addition, the method can be performed using a dsamplification product comprising topoisomerase recognition sites and,optionally, recombination sites, or cleavage products thereof, at ornear the 5′ terminus and the 3′ terminus of one or both ends, wherein,upon cleavage by the topoisomerases, the topoisomerase-charged nucleicacid molecule preferably contains a 5′ or 3′ overhang at one or bothends containing the topoisomerases. Once nucleic acid molecules arejoined by the methods described above, the resulting molecules may thenbe used in recombination reactions, such as those described elsewhereherein.

The first and second amplification products may be generated using twosets of PCR primer pairs. The two sets of PCR primer pairs may beselected such that, in the presence of an appropriate polymerase such asTaq polymerase and a template comprising the sequences to be amplified,the primers amplify portions of a target DNA sequence that are upstreamof and adjacent to, and downstream of and adjacent to, the site forinsertion of the selectable marker. In addition, the sets of PCR primerpairs may be designed such that the amplification products contain atopoisomerase recognition site and, following cleavage by the sitespecific topoisomerase, a 5′ overhanging sequence at the end to becovalently linked to the selectable marker. As such, the first PCRprimer pair includes 1) a first primer, which comprises, in anorientation from 5′ to 3′, a nucleotide sequence complementary to a 5′overhanging sequence of the end of the selectable marker to which theamplification product is to be covalently linked, a nucleotide sequencecomplementary to a topoisomerase recognition site, such that PCRintroduces a functional recognition site in the opposite strand (seeprimer sequences in FIG. 9D), and a nucleotide sequence complementary toa 3′ sequence of a target DNA sequence upstream of the insertion site;and 2) a second primer, which comprises a nucleotide sequence of thetarget genomic DNA upstream of the 3′ sequence to which the first primeris complementary, i.e., downstream of the insertion site. The second PCRprimer pair includes 1) a first primer, which comprises, from 5′ to 3′,a nucleotide sequence complementary to the 5′ overhanging sequence ofthe end of the selectable marker to which it is to be covalently linked,a nucleotide sequence complementary to a topoisomerase recognition site,such that PCR introduces a functional recognition site in the oppositestrand (see primer sequences in FIG. 9D), and a nucleotide sequence of a5′ sequence of a target DNA sequence, wherein the 5′ sequence of thetarget genomic DNA is downstream of the 3′ sequence of the target DNAsequence to which the first primer of the first PCR primer pair iscomplementary; and the second primer of the second primer pair comprisesa nucleotide sequence complementary to a 3′ sequence of the target DNAsequence that is downstream of the 5′ sequence of the target genomic DNAcontained in the first primer. The skilled artisan will recognize thatthe sequences of the primer that are complementary to the target genomicDNA are selected based on the sequence of the target DNA. These primersmay further comprise one or more recombination sites.

Upon contact of the nucleic acid molecule comprising the selectablemarker, the first and second amplification products, and a topoisomerase(if the molecules are not topoisomerase-charged), a ds recombinantnucleic acid molecule covalently linked in both strands is generatedaccording to a method of the invention. The generated ds recombinantnucleic acid molecule can be further amplified, if desired, using PCRprimers that are specific for an upstream and downstream sequence of thetarget genomic DNA, thus ensuring that only functional constructs areamplified. The generated ds recombinant nucleic acid molecule is usefulfor performing homologous recombination in a genome, for example, toknock-out the function of a gene in a cell, or to confer a novelphenotype on the cell containing the generated recombinant nucleic acidmolecule. The method can further be used to produce a transgenicnon-human organism having the generated ds recombinant nucleic acidmolecule stably maintained in its genome.

A method of the invention also is useful for covalently linking, anadapter or linker sequence to one or both ends of a nucleic acidmolecule of interest, including to each of a plurality of nucleic acidmolecules. For example, where it is desired to put linkers on both endsof a first nucleic acid molecule, the method can be performed bycontacting a topoisomerase with a first nucleic acid molecule, which hasa topoisomerase recognition site, or cleavage product thereof, at one orboth 3′ or 5′ termini and which can include hydroxyl groups at both 5′termini and one or more recombination sites; and a second nucleic acidmolecule and at least a third double stranded nucleotide sequence, eachof which can include a topoisomerase recognition site, or cleavageproduct thereof at the appropriate 3′ or 5′ terminus and which can alsoinclude, where desirable, a 5′ hydroxyl group at the same terminus andone or more recombination sites. An appropriate terminus is the terminusto which the linker is to be covalently linked in at least one strand tothe first nucleotide sequence. In one embodiment, one or both linkersequences contain an overhanging sequence that is complementary to asequence at the 5′ terminus of the end of the first nucleic acidmolecule to which the linker is to be covalently linked, therebyfacilitating the initial association of the nucleotide sequences in theproper (predetermined) orientation (see, for example, FIG. 9 and Example1.B). In performing such a method, the linker sequences comprising thesecond and at least third nucleotide sequence can be the same ordifferent.

FIG. 14 shows one example of a process for preparing a nucleic acidmolecule containing a topoisomerase (e.g., a type IA topoisomerase)bound to the 5′ terminus of one end of the sequence, and wherein thesame end further comprise a 3′ overhang (see (4) in FIG. 14). In step A,a nucleotide sequence to be modified with topoisomerase is digested witha restriction enzyme that generates a “sticky” end. The restrictednucleotide sequence is then contacted in step B with a linear, singlestranded nucleotide sequence which contains a topoisomerase attached the5′ terminus and a ligase (e.g., a DNA ligase such as T4 DNA ligase). Thelinear, single stranded nucleotide sequence also contains a region atthe 3′ terminus which shares sufficient sequence complementarity to the“sticky” end generated by the restriction enzyme, such that the twomolecules will hybridize. Thus, in step B, the two nucleotide sequencesare ligated to each other. In step C, the product of the second step iscontacted with a third nucleotide sequence which shares sequencecomplementarity to portions of the linear, single stranded nucleic acidmolecule generated in step B, and a ligase. The product of step C, shownin (4), is a nucleic acid molecule containing a topoisomerase attachedto the 5′ terminus of one end and a 3′ overhang on the same end. It willbe recognized that numerous variations of the exemplified method arewithin the scope of the invention. For example, similar processes can beperformed to prepare nucleic acid molecules which comprise topoisomeraseattached to the 3′ terminus of one end or which have a 5′ overhang orare blunt ended at the end to which a topoisomerase is attached. Inanother example, the nucleotide sequence labeled number 3 in FIG. 14 canbe produced in the following manner: a nucleic acid molecule can bedigested with a restriction enzyme to generate a nucleic acid moleculewith a single-stranded 5′ overhang that includes a type IA topoisomeraserecognition site. The nucleic acid molecule with the single strandedoverhang can then be contacted with type IA topoisomerase to generate atype IA topoisomerase-charged nucleic acid molecule.

FIG. 15 shows two embodiments of the invention in which single strandedor double stranded DNA is covalently linked to single stranded RNA.Where single stranded DNA is joined to single stranded RNA, the 3′ endof the ribonucleotide sequence is covalently linked to the 5′ end of thedeoxyribonucleotide sequence. Where double stranded DNA is joined tosingle stranded RNA, the 3′ terminus of the ribonucleotide sequenceshares sufficient sequence complementarity to the 3′ overhang of thedeoxyribonucleotide sequence such that the two molecules hybridize. Asabove, the 3′ end of the ribonucleotide sequence is also covalentlylinked to the 5′ end of the deoxyribonucleotide sequence. As will berecognized, numerous variations of the above are within the scope of theinvention. For example, the RNA molecule can be double stranded. Inanother example, all of the nucleotide sequences can bedeoxyribonucleotide sequences and/or can comprise one or morerecombination sites.

The present invention provides a ds recombinant nucleic acid moleculehaving, or which can be made to have, a first end and a second end, eachend including a 5′ terminus and a 3′ terminus, wherein the moleculecomprises a site-specific type IA topoisomerase recognition site at ornear a 5′ terminus of the first end, the second end, or both the firstend and the second end. The ds recombinant nucleic acid molecule canfurther include a type IB topoisomerase recognition site at or near a 3′termini of an end that does not include a type IA topoisomeraserecognition site. The ds recombinant nucleic acid molecule can be avector.

The present invention further provides a topoisomerase-charged dsrecombinant nucleic acid molecule having a first end and a second end,each end having a 5′ terminus and a 3′ terminus, wherein a site-specifictype IA topoisomerase is bound at the 5′ terminus of the first end, thesecond end, or both the first end and the second end. For example, thetopoisomerase-charged ds recombinant nucleic acid molecule can include atype IA topoisomerase bound at the 5′ termini of each of the first andsecond ends. The topoisomerase-charged nucleic acid ds recombinantnucleic acid molecule can include a type IB topoisomerase bound at a 3′termini of an end not bound by a type IA topoisomerase. Thetopoisomerase-charged ds recombinant nucleic acid molecule can be avector.

Kits

The present invention also provides kits, which contain componentsuseful for conveniently practicing the methods of the invention. In oneembodiment, a kit of the invention contains a first nucleic acidmolecule, which encodes a polypeptide, particularly a selectable marker,and contains a topoisomerase recognition site at each end. Preferably,the first nucleotide sequence comprises a topoisomerase-activatednucleotide sequence. More preferably, the topoisomerase-charged firstnucleotide sequence comprises a 5′ overhanging sequence at each end, andmost preferably the 5′ overhanging sequences are different from eachother. Optionally, each of the 5′ termini comprises a 5′ hydroxyl group.

In addition, the kit can contain at least a nucleotide sequence (orcomplement thereof) comprising a regulatory element, which can be anupstream or downstream regulatory element, or other element, and whichcontains a topoisomerase recognition site at one or both ends.Preferably, the kit contains a plurality of nucleic acid molecules, eachcomprising a different regulatory element or other element, for example,a sequence encoding a tag or other detectable molecule or a cellcompartmentalization domain. The different elements can be differenttypes of a particular regulatory element, for example, constitutivepromoters, inducible promoters and tissue specific promoters, or can bedifferent types of elements including, for example, transcriptional andtranslational regulatory elements, epitope tags, and the like. Suchnucleic acid molecules can be topoisomerase-activated, and can contain5′ overhangs or 3′ overhangs that facilitate operatively covalentlylinking the elements in a predetermined orientation, particularly suchthat a polypeptide such as a selectable marker is expressible in vitroor in one or more cell types.

The kit also can contain primers, including first and second primers,such that a primer pair comprising a first and second primer can beselected and used to amplify a desired ds recombinant nucleic acidmolecule covalently linked in one or both strands, generated usingcomponents of the kit. For example, the primers can include firstprimers that are complementary to elements that generally are positionedat the 5′ end of a generated ds recombinant nucleic acid molecule, forexample, a portion of a nucleic acid molecule comprising a promoterelement, and second primers that are complementary to elements thatgenerally are positioned at the 3′ end of a generated ds recombinantnucleic acid molecule, for example, a portion of a nucleic acid moleculecomprising a transcription termination site or encoding an epitope tag.Depending on the elements selected from the kit for generating a dsrecombinant nucleic acid molecule covalently linked in both strands, theappropriate first and second primers can be selected and used to amplifya full length functional construct.

In another embodiment, a kit of the invention contains a plurality ofdifferent elements, each of which can comprise one or more recombinationsites and/or can be topoisomerase-activated at one or both ends, andeach of which can contain a 5′ overhanging sequence or a 3′ overhangingsequence or a combination thereof. The 5′ or 3′ overhanging sequencescan be unique to a particular element, or can be common to plurality ofrelated elements, for example, to a plurality of different promoterelement. Preferably, the 5′ overhanging sequences of elements aredesigned such that one or more elements can be operatively covalentlylinked to provide a useful function, for example, an element comprisinga Kozak sequence and an element comprising a translation start site canhave complementary 5′ overhangs such that the elements can beoperatively covalently linked according to a method of the invention.

The plurality of elements in the kit can comprise any elements,including transcription or translation regulatory elements; elementsrequired for replication of a nucleotide sequence in a bacterial,insect, yeast, or mammalian host cell; elements comprising recognitionsequences for site specific nucleic acid binding proteins such asrestriction endonucleases or recombinases; elements encoding expressibleproducts such as epitope tags or drug resistance genes; and the like. Assuch, a kit of the invention provides a convenient source of differentelements that can be selected depending, for example, on the particularcells that a construct generated according to a method of the inventionis to be introduced into or expressed in. The kit also can contain PCRprimers, including first and second primers, which can be combined asdescribed above to amplify a ds recombinant nucleic acid moleculecovalently linked in one or both strands, generated using the elementsof the kit. Optionally, the kit further contains a site specifictopoisomerase in an amount useful for covalently linking in at least onestrand, a first nucleic acid molecule comprising a topoisomeraserecognition site to a second (or other) nucleic acid molecule, which canoptionally be topoisomerase-activated nucleic acid molecules ornucleotide sequences that comprise a topoisomerase recognition site.

In still another embodiment, a kit of the invention contains a firstnucleic acid molecule, which encodes a selectable marker, and contains atopoisomerase recognition site and/or a recombination site at each end;a first and second PCR primer pair, which can produce a first and secondamplification products that can be covalently linked in one or bothstrands, to the first nucleic acid molecule in a predeterminedorientation according to a method of the invention. Such a generatedconstruct can be introduced into a cell and can incorporate into thegenome of the cell by homologous recombination in a site specificmanner, where it can be stably maintained and can express a heterologouspolypeptide in the cell or can knock-out a target gene function. Atarget gene to be knocked-out, for example, can be any gene for which atleast part of the sequence is known or can be readily determined and thefunction of which it is desired to disrupt, for example, an oncogene, agene involved in apoptosis, a gene encoding a serine/threonine or atyrosine kinase, or any other gene.

The first PCR primer pair in a kit of the invention useful forgenerating a ds recombinant nucleic acid molecule covalently linked inboth strands, includes a first primer that comprises, in an orientationfrom 5′ to 3′, a nucleotide sequence complementary to a 5′ overhangingsequence of a nucleic acid molecule to which it is to be covalentlylinked (for example, an end of the nucleic acid molecule encoding theselectable marker), a nucleotide sequence complementary to atopoisomerase recognition site, such that PCR introduces a functionalrecognition site in the opposite strand (see primer sequences in FIG.9D), and/or a recombination site, and a nucleotide sequencecomplementary to a 3′ sequence of the target DNA sequence. The first PCRprimer pair also includes a second primer that comprises a nucleotidesequence of the target DNA sequence upstream of the 3′ sequence to whichthe first primer is complementary.

The second PCR primer pair of a kit useful for generating a dsrecombinant nucleic acid molecule covalently linked in both strands,includes a first primer that comprises, from 5′ to 3′, a nucleotidesequence complementary to a 5′ overhanging sequence of a nucleic acidmolecule to which it is to be covalently linked, a nucleotide sequencecomplementary to a topoisomerase recognition, such that PCR introduces afunctional recognition site in the opposite strand (see primer sequencesin FIG. 9D), site and/or a recombination site, and a nucleotide sequenceof a 5′ sequence of the target DNA sequence, wherein the 5′ sequence ofthe target gene is downstream of the 3′ sequence of the target DNAsequence to which the first primer of the first primer pair iscomplementary. The second PCR primer pair also includes a second primerthat comprises a nucleotide sequence complementary to a 3′ sequence ofthe target gene that is downstream of the 5′ sequence of the target DNAsequence contained in the first primer.

In another embodiment, a kit of the invention useful for generating a dsrecombinant nucleic acid molecule covalently linked in both strandscontains a first nucleic acid molecule, which encodes a transcriptionactivation domain and comprises a topoisomerase recognition site, orcleavage product thereof, at a 3′ terminus; and a second nucleic acidmolecule, which encodes a DNA binding domain and comprises atopoisomerase recognition site and/or a recombination site, or cleavageproduct thereof, at a 3′ terminus. Upon cleavage by the site specifictopoisomerase, the first or second nucleic acid molecule can have a 5′overhang, or both sequences can have 5′ overhangs, which are the same orare different from each other. Where the nucleic acid molecules have a5′ overhang, the overhang generally is complementary to a nucleic acidmolecule to which first or second nucleic acid molecule is to becovalently linked according to a method of the invention. The kit alsocan contain one or a pair of adapters, linkers or the like, which cancomprise a topoisomerase recognition site, or cleavage product thereof,at one or both 3′ termini, and, optionally, a hydroxyl group at the sameterminus/termini. Such adapters, linkers, or the like are selected suchthat they contain a 5′ overhang that is complementary to one or theother of the two nucleic acid molecules described above and part of thekit.

Similarly, a kit of the invention can contain one or a pair of adapters,linkers or the like, which comprise a topoisomerase recognition siteand/or a recombination site, or cleavage product thereof, at one or both5′ termini, and, optionally, a hydroxyl group at the same terminus (ortermini). Such adapters, linkers, or the like are selected such thatthey contain a 3′ overhang that is complementary to one or the other ofthe two nucleic acid molecules described above and part of the kit. Inaddition, the kit can contain one or a pair of adapters, linkers or thelike, which comprise a topoisomerase recognition site, or cleavageproduct thereof, at one or both 5′ and/or 3′ termini, and, optionally, ahydroxyl group at the same terminus/termini.

Adapters, linkers, or the like generally are selected such that theycontain a 5′ and/or a 3′ overhang that is complementary to one or theother of the two nucleic acid molecules as disclosed herein and part ofthe kit. Such adapters, linkers, or the like can be joined to the endsof nucleic acid molecules that are to covalently linked to one or theother of the first or second nucleic acid molecules provided with thekit, thus facilitating the construction of chimeric polynucleotidesencoding the bait and prey polypeptides useful in a two hybrid assay.Such a kit also can contain a PCR primer or primer pair, which can beused to prepare an amplified plurality of nucleotide sequencescomprising a topoisomerase recognition site, or cleavage product thereof(see Example 1).

A PCR primer pair in a kit of the invention, which can be used forgenerating a ds recombinant nucleic acid molecule covalently linked inone strand, can include a first primer that comprises, in an orientationfrom 5′ to 3′, a nucleotide sequence of a 5′ overhanging sequence of anucleic acid molecule to which it is to be linked (for example, an endof the nucleic acid molecule encoding the selectable marker), atopoisomerase recognition site (e.g., a type IA or type II topoisomeraserecognition site) and, optionally, a recombination site, and anucleotide sequence complementary to a 5′ sequence of the target DNAsequence. The PCR primer pair also includes a second primer thatcomprises a nucleotide sequence of the target DNA sequence downstream ofthe 5′ sequence to which the first primer is complementary.

In another embodiment, a kit of the invention contains a first nucleicacid molecule, which encodes a transcription activation domain andcomprises a site-specific topoisomerase recognition site (e.g., a typeIA or a type II topoisomerase recognition site) and, optionally, arecombination site, or cleavage product thereof, at a 5′ terminus; and asecond nucleic acid molecule, which encodes a DNA binding domain andcomprises a site-specific topoisomerase recognition site (e.g., a typeIA or a type II topoisomerase recognition site), or cleavage productthereof, at a 5′ terminus. Upon cleavage by the site specifictopoisomerase, the first or second nucleic acid molecule can have a 3′overhang, or both sequences can have 3′ overhangs, which are the same orare different from each other. Where the nucleic acid molecules have a3′ overhang, the overhang generally is complementary to a nucleic acidmolecule to which first or second nucleic acid molecule is to be linkedaccording to a method of the invention. The kit also can contain one ora pair of adapters, linkers or the like, which may comprise asite-specific topoisomerase recognition site (e.g., a type IA or a typeII topoisomerase recognition site), a cleavage product thereof, and/or arecombination site, at one or both 5′ and/or 3′ termini and/or locatedinternally, and which can contain a 5′ overhang that is complementary toone or the other of the two nucleic acid molecules of the kit.

A ds recombinant nucleic acid molecule covalently linked in one or bothstrands, and generated according to a method of the invention, can beused for various purposes, including, for example, for expressing apolypeptide in a cell, for diagnosing or treating a pathologiccondition, or the like. As such, the present invention provides amedicament, which can be useful for treating a pathologic condition byexpressing a polypeptide in one or more cells or by expressing anantisense molecule, or the like. Such a ds recombinant nucleic acidmolecule can be provided to a cell by contacting the cell ex vivo, thenadministering the cell to the subject, such a method also allowing forselection and/or expansion of the cells containing the ds recombinantnucleic acid molecule prior to such administration, or can be provideddirectly to the subject. For administration to a living subject, the dsrecombinant nucleic acid molecule, which is covalently linked in one orboth strands, generally is formulated in a composition suitable foradministration to the subject. Thus, the invention provides compositionscontaining a ds recombinant nucleic acid molecule covalently linked inone or both strands, generated according to a method of the invention.As disclosed herein, such nucleic acid molecules are useful asmedicaments for treating a subject suffering from a pathologicalcondition.

A composition for administration generally is formulated using one ormore pharmaceutically acceptable carriers as well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil or injectable organic esters. A pharmaceuticallyacceptable carrier can contain physiologically acceptable compounds thatact, for example, to stabilize or to increase the absorption of theconjugate. Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the composition,which can be, for example, orally or parenterally such as intravenously,and by injection, intubation, or other such method known in the art. Acomposition of the invention also can contain a second reagent such as adiagnostic reagent, nutritional substance, toxin, or therapeutic agent,for example, a cancer chemotherapeutic agent.

The ds recombinant nucleic acid molecule covalently linked in one orboth strands, can be incorporated within an encapsulating material suchas into an oil-in-water emulsion, a microemulsion, micelle, mixedmicelle, liposome, microsphere or other polymer matrix (see, forexample, Gregoriadis, Liposome Technology, Vol. I (CRC Press, BocaRaton, Fla. 1984); Fraley, et al., Trends Biochem. Sci., 6:77 (1981),each of which is incorporated herein by reference). Liposomes, forexample, which consist of phospholipids or other lipids, are nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer. “Stealth” liposomes (see, forexample, U.S. Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each ofwhich is incorporated herein by reference) are an example of suchencapsulating materials particularly useful for preparing apharmaceutical composition, and other “masked” liposomes similarly canbe used, such liposomes extending the time that a nucleic acid moleculeremains in the circulation. Cationic liposomes, for example, also can bemodified with specific receptors or ligands (Morishita et al., J. Clin.Invest., 91:2580-2585 (1993), which is incorporated herein byreference). The nucleic acid molecule also can be introduced into a cellby complexing it with an adenovirus-polylysine complex (see, forexample, Michael et al., J. Biol. Chem. 268:6866-6869 (1993), which isincorporated herein by reference). Such compositions can be particularlyuseful for introducing a nucleic acid molecule into a cell in vivo or invitro, including ex vivo, wherein the cell containing the nucleic acidmolecule is administered back to the subject (see U.S. Pat. No.5,399,346, which is incorporated herein by reference). A nucleic acidmolecule generated according to a method of the invention also can beintroduced into a cell using a biolistic method (see, for example, Sykesand Johnston, supra, 1999).

Host Cells

The invention also relates to host cells, or derivatives thereof,comprising one or more of the nucleic acid molecules or vectors of theinvention, particularly those nucleic acid molecules and vectorsdescribed in detail herein. Representative host cells that may be usedaccording to this aspect of the invention include, but are not limitedto, bacterial cells, yeast cells, plant cells and animal cells, andderivatives thereof. Preferred bacterial host cells include Escherichiaspp. cells (particularly E. coli cells and most particularly E. colistrains DHI0B, Stbl2, DH5a, DB3, DB3.1 (preferably E. coli LIBRARYEFFICIENCY® DB3.PM Competent Cells; Invitrogen Corporation, Carlsbad,Calif.), DB4, DB5, JDP682 and ccdA-over (see U.S. application Ser. No.09/518,188, filed Mar. 2, 2000, and U.S. provisional Application No.60/475,004, filed Jun. 3, 2003, by Louis Leong et al., entitled “CellsResistant to Toxic Genes and Uses Thereof,” the disclosures of which areincorporated by reference herein in their entireties); Bacillus spp.cells (particularly B. subtilis and B. megaterium cells); Streptomycesspp. cells; Erwinia spp. cells; Klebsiella spp. cells; Serratia spp.cells (particularly S. marcessans cells); Pseudomonas spp. cells(particularly P. aeruginosa cells); and Salmonella spp. cells(particularly S. typhimurium and S. typhi cells). Preferred animal hostcells include insect cells (most particularly Drosophila melanogastercells, Spodoptera frugiperda Sf9 and Sf2! cells and TrichoplusaHigh-Five cells), nematode cells (particularly C. elegans cells), aviancells, amphibian cells (particularly Xenopus laevis cells), reptiliancells, and mammalian cells (most particularly NIB3T3, CHO, COS, VERO,BHK and human cells). Preferred yeast host cells include Saccharomycescerevisiae cells and Pichia pastoris cells. In addition, derivatives ofsuch host cells are suitable for use in accordance with the presentinvention. These and other suitable host cells are availablecommercially, for example from Invitrogen Corporation (Carlsbad,Calif.), American Type Culture Collection (Manassas, Va.), andAgricultural Research Culture Collection (NRRL; Peoria, Ill.).

Methods for introducing the nucleic acid molecules and/or vectors of theinvention into the host cells described herein, to produce host cellscomprising one or more of the nucleic acid molecules and/or vectors ofthe invention, will be familiar to those of ordinary skill in the art.For instance, the nucleic acid molecules and/or vectors of the inventionmay be introduced into host cells using well known techniques ofinfection, transduction, electroporation, transfection, andtransformation. The nucleic acid molecules and/or vectors of theinvention may be introduced alone or in conjunction with other thenucleic acid molecules and/or vectors and/or proteins, peptides or RNAs.Alternatively, the nucleic acid molecules and/or vectors of theinvention may be introduced into host cells as a precipitate, such as acalcium phosphate precipitate, or in a complex with a lipid.Electroporation also may be used to introduce the nucleic acid moleculesand/or vectors of the invention into a host. Likewise, such moleculesmay be introduced into chemically competent cells such as E. coli. Ifthe vector is a virus, it may be packaged in vitro or introduced into apackaging cell and the packaged virus may be transduced into cells.Hence, a wide variety of techniques suitable for introducing the nucleicacid molecules and/or vectors of the invention into cells in accordancewith this aspect of the invention are well known and routine to those ofskill in the art. Such techniques are reviewed at length, for example,in Sambrook, J., et al., Molecular Cloning, a Laboratory Manual, 2ndEd., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp.16.30-16.55 (1989), Watson, J. D., et al., Recombinant DNA, 2nd Ed., NewYork: W.H. Freeman and Co., pp. 213-234 (1992), and Winnacker, E.-L.,From Genes to Clones, New York: VCH Publishers (1987), which areillustrative of the many laboratory manuals that detail these techniquesand which are incorporated by reference herein in their entireties fortheir relevant disclosures.

Polymerases

Polymerases for use in the invention include but are not limited topolymerases (DNA and RNA polymerases), and reverse transcriptases. DNApolymerases include, but are not limited to, Thermus thermophilus (Tth)DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoganeopolitana (Tne) DNA polymerase, Thermotoga maritima(Tma) DNApolymerase, Thermococcus litoralis (Tli or VENT™) DNA polymerase,Pyrococcus furiosus (Pfi) DNA polymerase, DEEPVENT™ DNA polymerase,Pyrococcus woosii (Pwo) DNA polymerase, Pyrococcus sp KOD2 (KOD) DNApolymerase, Bacillus sterothermophilus (Bst) DNA polymerase, Bacilluscaldophilus (Bca) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNApolymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermusflavus (Tfl/Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase,Thermus brockianus (DYNAZYME™) DNA polymerase, Methanobacteriumthermoautotrophicum (Mth) DNA polymerase, mycobacterium DNA polymerase(Mtb, Mlep), E. coli pol I DNA polymerase, T5 DNA polymerase, T7 DNApolymerase, and generally pol I type DNA polymerases and mutants,variants and derivatives thereof. RNA polymerases such as T3, T5, T7 andSP6 and mutants, variants and derivatives thereof may also be used inaccordance with the invention.

The nucleic acid polymerases used in the present invention may bemesophilic or thermophilic, and are preferably thermophilic. Preferredmesophilic DNA polymerases include Pol I family of DNA polymerases (andtheir respective Klenow fragments) any of which may be isolated fromorganism such as E. coli, H. influenzae, D. radiodurans, H. pylori, C.aurantiacus, R. prowazekii, T. pallidum, Synechocystis sp., B. subtilis,L. lactis, S. pneumoniae, M tuberculosis, M. leprae, M. smegmatis,Bacteriophage L5, phi-C31, T7, T3, T5, SP01, SP₀₂, mitochondrial from S.cerevisiae MIP-1, and eukaryotic C. elegans, and D. melanogaster(Astatke, M. et al., 1998, J. Mol. Biol. 278, 147-165), pol III type DNApolymerase isolated from any sources, and mutants, derivatives orvariants thereof, and the like. Preferred thermostable DNA polymerasesthat may be used in the methods and compositions of the inventioninclude Taq, Tne, Tma, Pfu, KOD, Tfl, Tth, Stoffel fragment, VENT™ andDEEPVENT™ DNA polymerases, and mutants, variants and derivatives thereof(U.S. Pat. No. 5,436,149; U.S. Pat. No. 4,889,818; U.S. Pat. No.4,965,188; U.S. Pat. No. 5,079,352; U.S. Pat. No. 5,614,365; U.S. Pat.No. 5,374,553; U.S. Pat. No. 5,270,179; U.S. Pat. No. 5,047,342; U.S.Pat. No. 5,512,462; WO 92/06188; WO 92/06200; WO 96/10640; WO 97/09451;Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F. C., et al., PCR Meth.Appl. 2:275-287 (1993); Flaman, J.-M, et al., Nucl. Acids Res.22(15):3259-3260 (1994)).

Reverse transcriptases for use in this invention include any enzymehaving reverse transcriptase activity. Such enzymes include, but are notlimited to, retroviral reverse transcriptase, retrotransposon reversetranscriptase, hepatitis B reverse transcriptase, cauliflower mosaicvirus reverse transcriptase, bacterial reverse transcriptase, Tth DNApolymerase, Taq DNA polymerase (Saiki, R. K., et al., Science239:487-491 (1988); U.S. Pat. Nos. 4,889,818 and 4,965,188), Tne DNApolymerase (WO 96/10640 and WO 97/09451), Tma DNA polymerase (U.S. Pat.No. 5,374,553) and mutants, variants or derivatives thereof (see, e.g.,WO 97/09451 and WO 98/47912). Preferred enzymes for use in the inventioninclude those that have reduced, substantially reduced or eliminatedRNase H activity. By an enzyme “substantially reduced in RNase Hactivity” is meant that the enzyme has less than about 20%, morepreferably less than about 15%, 10% or 5%, and most preferably less thanabout 2%, of the RNase H activity of the corresponding wildtype or RNaseH⁺ enzyme such as wildtype Moloney Murine Leukemia Virus (M-MLV), AvianMyeloblastosis Virus (AMV) or Rous Sarcoma Virus (RSV) reversetranscriptases. The RNase H activity of any enzyme may be determined bya variety of assays, such as those described, for example, in U.S. Pat.No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265(1988) and in Gerard, G. F., et al., FOCUS 14(5):91 (1992), thedisclosures of all of which are fully incorporated herein by reference.Particularly preferred polypeptides for use in the invention include,but are not limited to, M-MLV H⁻ reverse transcriptase, RSV H⁻ reversetranscriptase, AMV H⁻ reverse transcriptase, RAV (rous-associated virus)H⁻ reverse transcriptase, MAV (myeloblastosis-associated virus) H⁻reverse transcriptase and HIV H⁻ reverse transcriptase. (See U.S. Pat.No. 5,244,797 and WO 98/47912). It will be understood by one of ordinaryskill, however, that any enzyme capable of producing a DNA molecule froma ribonucleic acid molecule (i.e., having reverse transcriptaseactivity) may be equivalently used in the compositions, methods and kitsof the invention.

The enzymes having polymerase activity for use in the invention may beobtained commercially, for example from Invitrogen Corporation(Carlsbad, Calif.), Perkin-Elmer (Branchburg, N.J.), New England BioLabs(Beverly, Mass.) or Boehringer Mannheim Biochemicals (Indianapolis,Ind.). Enzymes having reverse transcriptase activity for use in theinvention may be obtained commercially, for example from InvitrogenCorporation (Carlsbad, Calif.), Pharmacia (Piscataway, N.J.), Sigma(Saint Louis, Mo.) or Boehringer Mannheim Biochemicals (Indianapolis,Ind.). Alternatively, polymerases or reverse transcriptases havingpolymerase activity may be isolated from their natural viral orbacterial sources according to standard procedures for isolating andpurifying natural proteins that are well-known to one of ordinary skillin the art (see, e.g., Houts, G. E., et al., J. Virol. 29:517 (1979)).In addition, such polymerases/reverse transcriptases may be prepared byrecombinant DNA techniques that are familiar to one of ordinary skill inthe art (see, e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265(1988); U.S. Pat. No. 5,244,797; WO 98/47912; Soltis, D. A., and Skalka,A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376 (1988)). Examples ofenzymes having polymerase activity and reverse transcriptase activitymay include any of those described in the present application.

Methods of Nucleic Acid Synthesis, Amplification and Sequencing

The present invention may be used in combination with any methodinvolving the synthesis of nucleic acid molecules, such as DNA(including cDNA) and RNA molecules. Such methods include, but are notlimited to, nucleic acid synthesis methods, nucleic acid amplificationmethods and nucleic acid sequencing methods. Such methods may be used toprepare molecules (e.g., starting molecules) used in the invention or tofurther manipulate molecules or vectors produced by the invention.

Nucleic acid synthesis methods according to this aspect of the inventionmay comprise one or more steps. For example, the invention provides amethod for synthesizing a nucleic acid molecule comprising (a) mixing anucleic acid template (e.g., a nucleic acid molecules or vectors of theinvention) with one or more primers and one or more enzymes havingpolymerase or reverse transcriptase activity to form a mixture; and (b)incubating the mixture under conditions sufficient to make a firstnucleic acid molecule complementary to all or a portion of the template.According to this aspect of the invention, the nucleic acid template maybe a DNA molecule such as a cDNA molecule or library, or an RNA moleculesuch as a mRNA molecule. Conditions sufficient to allow synthesis suchas pH, temperature, ionic strength, and incubation times may beoptimized by those skilled in the art. If desired, recombination sitesand/or topoisomerase recognition sites may be added to such synthesizedmolecules during or after the synthesis process (see for sample, U.S.patent application Ser. No. 09/177,387 filed Oct. 23, 1998 based on U.S.provisional patent application No. 60/065,930 filed Oct. 24, 1997).

In accordance with the invention, the target or template nucleic acidmolecules or libraries may be prepared from nucleic acid moleculesobtained from natural sources, such as a variety of cells, tissues,organs or organisms. Cells that may be used as sources of nucleic acidmolecules may be prokaryotic (bacterial cells, including those ofspecies of the genera Escherichia, Bacillus, Serratia, Salmonella,Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria,Treponema, Mycoplasma, Borrelia, Legionella, Pseudomonas, Mycobacterium,Helicobacter, Erwinia, Agrobacterium, Rhizobium, and Streptomyces) oreukaryotic (including fungi (especially yeast's), plants, protozoans andother parasites, and animals including insects (particularly Drosophilaspp. cells), nematodes (particularly Caenorhabditis elegans cells), andmammals (particularly hurrian cells)).

Of course, other techniques of nucleic acid synthesis which may beadvantageously used will be readily apparent to one of ordinary skill inthe art.

In other aspects of the invention, the invention may be used incombination with methods for amplifying or sequencing nucleic acidmolecules. Nucleic acid amplification methods according to this aspectof the invention may include the use of one or more polypeptides havingreverse transcriptase activity, in methods generally known in the art asone-step (e.g., one-step RT-PCR) or two-step (e.g., two-step RT-PCR)reverse transcriptase-amplification reactions. For amplification of longnucleic acid molecules (i.e., greater than about 3-5 Kb in length), acombination of DNA polymerases may be used, as described in WO 98/06736and WO 95/16028.

Amplification methods according to the invention may comprise one ormore steps. For example, the invention provides a method for amplifyinga nucleic acid molecule comprising (a) mixing one or more enzymes withpolymerase activity with one or more nucleic acid templates; and (b)incubating the mixture under conditions sufficient to allow the enzymewith polymerase activity to amplify one or more nucleic acid moleculescomplementary to all or a portion of the templates. The invention alsoprovides nucleic acid molecules amplified by such methods. If desired,recombination sites may be added to such amplified molecules during orafter the amplification process (see for example, U.S. patentapplication Ser. No. 09/177,387 filed Oct. 23, 1998, based on U.S.provisional patent application No. 60/065,930 filed Oct. 24, 1997, thedisclosures of which are incorporated herein by reference in theirentireties).

General methods for amplification and analysis of nucleic acid moleculesor fragments are well known to one of ordinary skill in the art (see,e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159; Innis, M. A.,et al., eds., PCR Protocols: A Guide to Methods and Applications, SanDiego, Calif.: Academic Press, Inc. (1990); Griffin, H. G., and Griffin,A. M., eds., PCR Technology: Current Innovations, Boca Raton, Fla.: CRCPress (1994)). For example, amplification methods which may be used inaccordance with the present invention include PCR (U.S. Pat. Nos.4,683,195 and 4,683,202), Strand Displacement Amplification (SDA; U.S.Pat. No. 5,455,166; EP 0 684 315), and Nucleic Acid Sequence-BasedAmplification (NASBA; U.S. Pat. No. 5,409,818; EP 0 329 822).

Typically, these amplification methods comprise: (a) mixing one or moreenzymes with polymerase activity with the nucleic acid sample in thepresence of one or more primer sequences, and (b) amplifying the nucleicacid sample to generate a collection of amplified nucleic acidfragments, preferably by PCR or equivalent automated amplificationtechnique.

Following amplification or synthesis by the methods of the presentinvention, the amplified or synthesized nucleic acid fragments may beisolated for further use or characterization. This step is usuallyaccomplished by separation of the amplified or synthesized nucleic acidfragments by size or by any physical or biochemical means including gelelectrophoresis, capillary electrophoresis, chromatography (includingsizing, affinity and immunochromatography), density gradientcentrifugation and immunoadsorption. Separation of nucleic acidfragments by gel electrophoresis is particularly preferred, as itprovides a rapid and highly reproducible means of sensitive separationof a multitude of nucleic acid fragments, and permits direct,simultaneous comparison of the fragments in several samples of nucleicacids. One can extend this approach, in another preferred embodiment, toisolate and characterize these fragments or any nucleic acid fragmentamplified or synthesized by the methods of the invention. Thus, theinvention is also directed to isolated nucleic acid molecules producedby the amplification or synthesis methods of the invention.

In this embodiment, one or more of the amplified or synthesized nucleicacid fragments are removed from the gel which was used foridentification (see above), according to standard techniques such aselectroelution or physical excision. The isolated unique nucleic acidfragments may then be inserted into standard vectors, includingexpression vectors, suitable for transfection or transformation of avariety of prokaryotic (bacterial) or eukaryotic (yeast, plant or animalincluding human and other mammalian) cells. Alternatively, nucleic acidmolecules produced by the methods of the invention may be furthercharacterized, for example by sequencing (i.e., determining thenucleotide sequence of the nucleic acid fragments), by methods describedbelow and others that are standard in the art (see, e.g., U.S. Pat. Nos.4,962,022 and 5,498,523, which are directed to methods of DNAsequencing).

Nucleic acid sequencing methods according to the invention may compriseone or more steps. For example, the invention may be combined with amethod for sequencing a nucleic acid molecule comprising (a) mixing anenzyme with polymerase activity with a nucleic acid molecule to besequenced, one or more primers, one or more nucleotides, and one or moreterminating agents (such as a dideoxynucleotides) to form a mixture; (b)incubating the mixture under conditions sufficient to synthesize apopulation of molecules complementary to all or a portion of themolecule to be sequenced; and (c) separating the population to determinethe nucleotide sequence of all or a portion of the molecule to besequenced.

Nucleic acid sequencing techniques which may be employed include dideoxysequencing methods such as those disclosed in U.S. Pat. Nos. 4,962,022and 5,498,523.

Kits

In another aspect, the invention provides kits which may be used inconjunction with the invention. Kits of the invention may contain anynumber of components but typically will contain at least two components.Kits according to this aspect of the invention may comprise one or morecontainers, which may contain one or more components selected from thegroup consisting of one or more nucleic acid molecules or vectors of theinvention, one or more primers, the molecules and/or compounds of theinvention, supports of the invention, one or more polymerases, one ormore reverse transcriptases, one or more recombination proteins (orother enzymes for carrying out the methods of the invention), one ormore topoisomerases, one or more buffers, one or more detergents, one ormore restriction endonucleases, one or more nucleotides, one or moreterminating agents (e.g., ddNTPs), one or more transfection reagents,pyrophosphatase, and the like. The kits of the invention may alsocomprise instructions for carrying out methods of the invention.

For example, a kit of the invention may comprise (1) a first nucleicacid molecule which comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, etc.) recombination sites and/or one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, etc.) toposiomerase recognition sites and (2)instructions for covalently linking the first nucleic molecule toanother nucleic acid molecule using methods described herein. Inparticular embodiments, the instructions describe methods for linkingtwo or more nucleic molecules in either one or both strands. In arelated embodiment, the first nucleic acid molecule is topoisomeraseadapted prior to inclusion in the kit.

Additional kits of the invention can contain, for example, one or moretopoisomerase-charged nucleic acid molecule substrates, which caninclude one or more control nucleic acid sequences which can be useful,for example, to test the accuracy or fidelity of the components of thekit; one or more topoisomerases; one or more compositions comprising oneor more topoisomerases; one or more recombinases (or recombinationproteins); one or more compositions comprising one or more recombinases(or recombination proteins); one or more primers, which can comprise atleast one topoisomerase recognition site and/or at least onerecombination site, a nucleotide sequence complementary to at least onetopoisomerase recognition site and/or at least one recombination site,or both at least one topoisomerase recognition site and at least onenucleotide sequence complementary to at least one topoisomeraserecognition site; one or more cells, which can contain or be useful forcontaining a nucleic acid molecule of the kit or generated using thekit; one or more reagents, polymers, buffers, or the like, forperforming a method using the kit; instructions for performing a methodusing the kit; and the like.

In another aspect, a kit of the invention may contain a nucleic acidmolecule having a first end and a second end, and encoding a polypeptideto be expressed, for example, a selectable marker, wherein the nucleicacid molecule comprises a topoisomerase recognition site or cleavageproduct thereof at the 3′ terminus of one or both ends. Optionally, thenucleic acid molecule contains a hydroxyl group at the 5′ terminus ofone or both of the other ends, i.e., at the ends that do not contain atopoisomerase recognition site or that are not topoisomerase-charged.Further, one or both 5′ termini may comprise overhanging sequences,which are different from each other. A kit of the invention also cancontain a nucleic acid molecule having a first end and a second end, andencoding a polypeptide to be expressed, for example, a selectablemarker, wherein the nucleic acid molecule comprises a topoisomeraserecognition site or cleavage product thereof at the 5′ terminus of oneor both ends. Optionally, the nucleic acid molecule contains a hydroxylgroup at the 3′ terminus of one or both ends, and preferably, one orboth 3′ termini comprise overhanging sequences, which are different fromeach other. In addition, a kit of the invention can contain a nucleicacid molecule having a first end and a second end, and encoding apolypeptide to be expressed, for example, a selectable marker, whereinthe nucleic acid molecule comprises a topoisomerase recognition site orcleavage product thereof at the 5′ terminus and the 3′ terminus of oneor both ends. As such, it should be recognized that a kit of theinvention can include any of various combinations of such nucleic acidmolecules comprising one or more topoisomerase recognition sites ortopoisomerase-charged nucleic acid molecules.

A kit of the invention also can contain a nucleic acid moleculecomprising a regulatory element or other nucleotide sequence, forexample, a coding sequence, and a topoisomerase recognition site and/ora recombination site, or cleavage product thereof, at a 3′ terminus ofat least a first end and, optionally, a hydroxyl group at the 5′terminus of an end containing the recognition site; or comprising atopoisomerase recognition site or cleavage product thereof at a 5′terminus of at least a first end, and, optionally, a hydroxyl group atthe 3′ terminus of the end containing the recognition site; orcomprising a topoisomerase recognition site at the 5′ terminus and 3′terminus of at least a first end. In certain embodiments, the kit maycontain a variety of upstream regulatory elements, a variety ofdownstream regulatory elements, a variety of elements useful detectingor identifying a molecule containing the element, and combinationsthereof. For example, the kit can contain a variety of gene promoterelements, which are active constitutively or inducibly and in a few ormany different types of cells, elements that permit ribosome bindingsuch as an internal ribosome entry site, an element encoding a Kozaksequence or an initiator methionine, or the like. In addition, oralternatively, the kit can contain a variety of downstream regulatoryelements such a polyadenylation signal sequences, sequences thatterminate transcription or translation, or the like. Similarly, the kitcan contain elements encoding detectable markers such as epitope tags,or the like. In certain such aspects of the invention, the kit containsa variety of such elements, each of which contains at least onetopoisomerase recognition site and/or at least one recombination site.In certain other such aspects, these elements may contain an overhangingsequence such that they can be operably covalently linked to each otheror to a nucleic acid molecule encoding a polypeptide such as aselectable marker according to a method of the invention.

Optionally, the kit contains element specific primers, which can amplifya construct containing one of the variety of elements included in thekit. Where the kit contains such primers, the nucleic acid moleculescomprising the regulatory or other element has a nucleotide sequencethat can be specifically recognized by the primer and that results inextension of the primer through and including the regulatory element. Inparticular, the kit can contain element specific forward and reverseprimers, which can be combined to produce a primer pair that amplifies,for example, a construct containing a particular 5′ regulatory elementand a particular 3′ regulatory element of the kit. Such a primer paircan selectively amplify a desired functional covalently linked dsnucleic acid molecule generated according to a method of the invention,but does not amplify partial reaction products.

In another embodiment, a kit of the invention contains a first nucleicacid molecule, which has a first end and a second end, contains atopoisomerase recognition site, or cleavage product thereof, and/or arecombination site, at or near one or both 3′ termini, and encodes atranscription activation domain; and a second nucleic acid molecule,which has a first end and a second end, contains a topoisomeraserecognition site, or cleavage product thereof, at or near one or both 3′termini, and encodes a DNA binding domain; or contains a first nucleicacid molecule, which has a first end and a second end, contains atopoisomerase recognition site, or cleavage product thereof, and/or arecombination site, at or near one or both 5′ termini, and encodes atranscription activation domain; and a second nucleic acid molecule,which has a first end and a second end, contains a topoisomeraserecognition site, or cleavage product thereof, and/or a recombinationsite, at or near one or both 5′ termini, and encodes a DNA bindingdomain. A kit of the invention also can contain a first nucleic acidmolecule, which has a first end and a second end, and encodes atranscription activation domain, and a second nucleic acid molecule,which has a first end and a second end, and encodes a DNA bindingdomain, wherein at least the first nucleic acid molecule—or the secondnucleic acid molecule contains a topoisomerase recognition site, orcleavage product thereof, at or near a 5′ terminus and at or near 3′terminus of at least one end, and wherein the other ds nucleotidecontains a 3′ hydrdxyl and 5′ hydroxyl at the end to be covalentlylinked to the end of the nucleic acid molecule comprising therecognition sites. Such a kit is useful, for example, for generatingcovalently linked ds recombinant nucleic acid molecules encodingchimeric polypeptides for performing a two hybrid assay. The kit canfurther contain a primer pair, which can amplify a nucleotide sequenceto be operably linked to the first or second nucleic acid molecule,wherein at least one primer of the primer pair comprises a topoisomeraserecognition site, a complement of a topoisomerase recognition site, orboth. Preferably, an amplification product generated using such a primerpair contains, following cleavage by a site-specific topoisomerase, a 3′or 5′ overhanging sequence that is complementary to the first or secondnucleic acid molecule to which it is to be covalently linked. Such a kitcan facilitate the generation of recombinant polynucleotides thatcomprise a first or second nucleotide sequence of the kit and encode achimeric polypeptide useful for performing a two hybrid assay.

The present invention also relates to additional kits for carrying outthe methods of the invention, and particularly for use in creating theproduct nucleic acid molecules of the invention. The invention alsorelates to kits for carrying out homologous recombination (particularlygene targeting) according to the methods of the invention. Such kits ofthe invention may also comprise further components for furthermanipulating the recombination site-containing molecules and/orcompounds produced by the methods of the invention. The kits of theinvention may comprise one or more nucleic acid molecules of theinvention (particularly starting molecules comprising one or morerecombination sites and optionally comprising one or more reactivefunctional moieties), one or more molecules and/or compounds of theinvention, one or more supports of the invention and/or one or morevectors of the invention. Such kits may optionally comprise one or moreadditional components selected from the group consisting of one or morehost cells or derivatives thereof, one or more nucleotides, one or morepolymerases and/or reverse transcriptases, one or more suitable buffers,one or more primers, one or more terminating agents, one or morepopulations of molecules for creating combinatorial libraries and one ormore combinatorial libraries.

In another embodiment, a kit of the invention contains a first nucleicacid molecule, which encodes a polypeptide, particularly a selectablemarker, and contains a topoisomerase recognition site at each end. Incertain preferred such embodiments, the first nucleic acid molecule is acircular molecule (for example, a plasmid, vector, etc.) and comprisesat least one recombination site, and more preferably at least tworecombination sites, flanking the one or more, preferably two or more,topoisomerase recognition sites on the molecule. Preferably, the firstnucleotide sequence comprises a topoisomerase-activated nucleotidesequence. More preferably, the topoisomerase-charged first nucleotidesequence comprises a 5′ overhanging sequence at each end, and mostpreferably the 5′ overhanging sequences are different from each other.Optionally, each of the 5′ termini comprises a 5′ hydroxyl group.

Kits according to this aspect of the invention may also contain at leasta nucleotide sequence comprising a regulatory element, which can be anupstream or downstream regulatory element, or other element, whichcontains one or more topoisomerase recognition sites and, optionally,contains one or more recombination sites at one or both ends.Preferably, the kit contains a plurality of nucleic acid molecules, eachcomprising a different regulatory element or other element, for example,a sequence encoding a tag or other detectable molecule or a cellcompartmentalization domain. The different elements can be differenttypes of a particular regulatory element, for example, constitutive orinducible promoters or tissue specific promoters, or can be differenttypes of elements including, for example, transcriptional andtranslational regulatory elements, epitope tags, and the like. Suchnucleic acid molecules can be topoisomerase-activated, and can contain5′ overhanging sequences that facilitate operably covalently linking theelements in a predetermined orientation, particularly such that apolypeptide such as a selectable marker is expressible in vitro or inone or more cell types.

Such kits also may contain primers, including first and second primers,such that a primer pair comprising a first and second primer can beselected and used to amplify a desired covalently linked ds recombinantnucleic acid molecule generated using components of the kit. Forexample, the primers can include first primers that are complementary toelements that generally are positioned at the 5′ end of a generated dsrecombinant nucleic acid molecule, for example, a portion of a nucleicacid molecule comprising a promoter element, and second primers that arecomplementary to elements that generally are positioned at the 3′ end ofa generated ds recombinant nucleic acid molecule, for example, a portionof a nucleic acid molecule comprising a transcription termination siteor encoding an epitope tag. Depending on the elements selected from thekit for generating a covalently linked ds recombinant nucleic acidmolecule, the appropriate first and second primers can be selected andused to amplify a full length functional construct.

In another embodiment, a kit of the invention contains a plurality ofdifferent elements, each of which can be topoisomerase-activated at oneor both ends, and each of which can contain a 5′ overhanging sequence.The 5′ overhanging sequences can be unique to a particular element, orcan be common to plurality of related elements, for example, to aplurality of different promoter element. Preferably, the 5′ overhangingsequences of elements are designed such that one or more elements can beoperably covalently linked to provide a useful function, for example, anelement comprising a Kozak sequence and an element comprising atranslation start site can have complementary 5′ overhangs such that theelements can be operably covalently linked according to a method of theinvention.

The plurality of elements in the kit can comprise any elements,including transcription or translation regulatory elements; elementsrequired for replication of a nucleotide sequence in a bacterial,insect, yeast, or mammalian host cell; elements comprising recognitionsequences for site specific nucleic acid binding proteins such asrestriction endonucleases or recombinases; elements encoding expressibleproducts such as epitope tags or drug resistance genes; and the like. Assuch, a kit of the invention provides a convenient source of differentelements that can be selected depending, for example, on the particularcells that a construct generated according to a method of the inventionis to be introduced into or expressed in. The kit also can contain PCRprimers, including first and second primers, which can be combined asdescribed above to amplify a covalently linked ds recombinant nucleicacid molecule generated using the elements of the kit. Optionally, thekit further contains one or more topoisomerases (e.g., one or moresite-specific topoisomerases) and/or one or more recombinases (orrecombination proteins) in an amount useful for covalently linking afirst nucleic acid molecule comprising a topoisomerase recognition siteto a second (or other) nucleic acid molecule, which can betopoisomerase-activated nucleic acid molecules or can be nucleotidesequences that comprise a topoisomerase recognition site.

In still another embodiment, a kit of the invention contains a firstnucleic acid molecule, which encodes a selectable marker, and contains atopoisomerase recognition site at each end; a first and second PCRprimer pair, which can produce a first and second amplification productsthat can be covalently linked to the first nucleic acid molecule in apredetermined orientation according to a method of the invention. Such agenerated construct can be introduced into a cell and can incorporateinto the genome of the cell by homologous recombination in a sitespecific manner, where it can be stably maintained and can express aheterologous polypeptide in the cell or can knock-out a target genefunction. A target gene to be knocked-out, for example, can be any genefor which at least part of the sequence is known or can be readilydetermined and the function of which it is desired to disrupt, forexample, an oncogene, a gene involved in apoptosis, a gene encoding aserine/threonine or a tyrosine kinase, or any other gene.

The first PCR primer pair in a kit of the invention includes a firstprimer that comprises, in an orientation from 5′ to 3′, a nucleotidesequence complementary to a 5′ overhanging sequence of a nucleic acidmolecule to which it is to be covalently linked (for example, an end ofthe nucleic acid molecule encoding the selectable marker), a nucleotidesequence complementary to a topoisomerase recognition site, such thatPCR introduces a functional recognition site in the opposite strand (seeprimer sequences in FIG. 9D), and/or to a recombination site, and anucleotide sequence complementary to a 3′ sequence of the target DNAsequence. The first PCR primer pair also includes a second primer thatcomprises a nucleotide sequence of the target DNA sequence upstream ofthe 3′ sequence to which the first primer is complementary.

The second PCR primer pair of a kit of the invention includes a firstprimer that comprises, from 5′ to 3′, a nucleotide sequencecomplementary to a 5′ overhanging sequence of a nucleic acid molecule towhich it is to be covalently linked, a nucleotide sequence complementaryto a topoisomerase recognition site, such that PCR introduces afunctional recognition site in the opposite strand (see primer sequencesin FIG. 9D), and optionally, a nucleotide sequence complementary to arecombination site, and a nucleotide sequence of a 5′ sequence of thetarget DNA sequence, wherein the 5′ sequence of the target gene isdownstream of the 3′ sequence of the target DNA sequence to which thefirst primer of the first primer pair is complementary. The second PCRprimer pair also includes a second primer that comprises a nucleotidesequence complementary to a 3′ sequence of the target gene that isdownstream of the 5′ sequence of the target DNA sequence contained inthe first primer.

In another embodiment, a kit of the invention contains a first nucleicacid molecule, which encodes a transcription activation domain andcomprises a topoisomerase recognition site, or cleavage product thereof,at or near a 3′ terminus; and a second nucleic acid molecule, whichencodes a DNA binding domain and comprises a topoisomerase recognitionsite and optionally a recombination site, or cleavage product thereof,at or near a 3′ terminus. Upon cleavage by the site specifictopoisomerase, the first or second nucleic acid molecule can have a 5′overhang, or both sequences can have 5′ overhangs, which are the same orare different from each other. Where the nucleic acid molecules have a5′ overhang, the overhang generally is complementary to a nucleic acidmolecule to which first or second nucleic acid molecule is to becovalently linked according to a method of the invention.

The kit also can contain one or a pair of adapters, linkers or the like,which comprise a topoisomerase recognition site and, optionally, arecombination site, or cleavage product thereof, at one or both 3′termini, and, optionally, a hydroxyl group at the same terminus/termini.Such adapters, linkers, or the like are selected such that they containa 5′ overhang that is complementary to one or the other of the twonucleic acid molecules described above and part of the kit. Similarly,the kit also can contain one or a pair of adapters, linkers or the like,which comprise a topoisomerase recognition site and, optionally, arecombination site, or cleavage product thereof, at one or both 5′termini, and, optionally, a hydroxyl group at the same terminus/termini.Such adapters, linkers, or the like are selected such that they containa 3′ overhang that is complementary to one or the other of the twonucleic acid molecules described above and part of the kit. In addition,the kit can contain one or a pair of adapters, linkers or the like,which comprise a topoisomerase recognition site, or cleavage productthereof, at or near one or both 5′ and/or 3′ termini, and, optionally, ahydroxyl group at the same terminus/termini. Such adapters, linkers, orthe like are selected such that they contain a 5′ and/or a 3′ overhangthat is complementary to one or the other of the two nucleic acidmolecules described above and part of the kit. Such adapters, linkers,or the like can be joined to the ends of nucleic acid molecules that areto covalently linked to one or the other of the first or second nucleicacid molecules provided with the kit, thus facilitating the constructionof chimeric polynucleotides encoding the bait and prey polypeptidesuseful in a two hybrid assay. Such a kit also can contain a PCR primeror primer pair, which can be used to prepare an amplified plurality ofnucleotide sequences comprising a topoisomerase recognition site, orcleavage product thereof. Additional kits according to this aspect ofthe invention may optionally comprise one or more additional componentssuch as one or more topoisomerases, one or more recombination proteins,one or more vectors, one or more polypeptides having polymeraseactivity, and one or more host cells.

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods andapplications described herein are readily apparent from the descriptionof the invention contained herein in view of information known to theordinarily skilled artisan, and may be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES Example 1 Construction of Covalently Linked Double StrandedRecombinant Nucleic Acid Molecules Using Topoisomerase

This example demonstrates that topoisomerase can be used to producecovalently linked double stranded (ds) recombinant nucleic acidmolecules.

A. Methods

Except where indicated, studies were performed using the followingmethods. PCR was performed in 50 μl reactions, including 10 ng plasmid(template), 100 ng each primer, 2.5 Units Taq DNA polymerase (Sigma), 5μl 10×PCR buffer, and 4 μl of dNTPs (200 μM each). An initialdenaturation was performed by incubating the reaction at 94° C. for 4min; followed by 30 cycles of PCR using 94° C. (45 sec) fordenaturation, 55° C. (45 sec) for primer annealing and 72° C. (1 min perkb of target sequence) for extension. After cycling, the reactions wereincubated at 72° C. (10 min), and then placed at 4° C.

Topoisomerase joining reactions were performed in 5 μl, including 50-100ng each amplified element (PCR-generated or synthetic), 0.5 μl 500 mMTris (pH 7.5), and 0.5 μg topoisomerase. Reactions were incubated atroom temperature for 5 min, then 1-2 μl of the Topo-linked product wasused for linear fragment generation.

Linear fragment generation by PCR was performed in 50 μl reactions,including 1-2 μl of the Topo-linked product (template), 100 ng eachprimer, 2.5 U Taq DNA polymerase (Sigma), 5 μl 10×PCR buffer, and 4 μldNTPs (200 μM each). PCR was performed as described above.

The resultant linear fragment was purified using a SNAP Miniprep Kit(Invitrogen Corporation, Carlsbad, Calif.) as described by themanufacturer. Essentially, 100 μl PCR product was mixed with 300 μlBinding Buffer; 750 μl isopropanol, and the mixture was applied to aSNAP Miniprep Column/Collection Tube and centrifuged at 7,000 rpm for 30sec. The column was washed with 700 μl Wash Buffer, centrifuged at 7,000rpm for 30 sec; then washed with 900 μl 1× Final Wash and centrifuged at7,000 rpm for 30 sec. The column was then centrifuged at 7,000 rpm foran additional 30 sec to remove all remaining liquid. Water (30 to 50 μl)was added and the column was centrifuged at 7,000 rpm for 30 sec toelute the purified DNA. DNA concentration was determined byspectrophotometry.

B. Generation of Topoisomerase Linked Linear Nucleic Acid Molecules

PCR primers were designed to examine the directional addition ofelements to the coding sequence of green fluorescent protein (GFP; seeFIG. 9A-C). The CMV promoter (approximately 700 bp) and BGHpolyadenylation signal sequence (approximately 380 bp) were amplifiedfrom a pCMV/myc/nuc plasmid template, and the GFP element (approximately700 bp) was amplified from a pcDNA3.1/GFP plasmid template (InvitrogenCorporation, Carlsbad, Calif.) using the primers indicated in FIG. 9D.The resultant amplification products were joined using topoisomerase asdescribed above, and a portion of the ligation reaction was used astemplate for PCR with primers F6945 (SEQ ID NO: 11) and F6948 (SEQ IDNO: 15) to amplify the entire construct (CMV+GFP+BGH; approximately1,700 bp). In addition, 5 μl of the ligation mixture was treated withproteinase K for 30 min at 37° C. to remove any bound topoisomerase, andthen subjected to electrophoresis on a 3-8% NuPAGE Tris-acetate gel toexamine the ligated products.

Only a small amount of ligation product of the correct size (1.7 kb) wasobserved when the recombinant nucleic acid molecules were generatedusing elements having palindromic overhanging sequence (FIG. 9A or 9B),whereas significant quantities of the desired product were generatedusing elements having non-palindromic overhangs (FIG. 9C). These resultsdemonstrate that the efficiency of generating ds recombinant nucleicacid molecule covalently linked in both strands containing nucleotidesequences operatively linked in a predetermined orientation is relatedto the nature of the overhang sequence. In particular, the selection ofoverhanging sequences that lack palindromic regions result in theefficient generation of a desired ds recombinant nucleic acid moleculecovalently linked in both strands, whereas the presence of palindromicsequences in the overhangs allows the formation of ligation productsother than the intended product, thus decreasing the efficiency ofgenerating a desired product.

Example 2 Functional Characterization of Topoisomerase-Generated dsRecombinant Nucleic Acid Molecules

This example demonstrates that a method of the invention provides ameans to generate functional ds recombinant nucleic acid moleculescovalently linked in both strands.

A. Expression of Sense and Antisense mRNA from a Topo-Ligated Construct

The ability to create a ds recombinant nucleic acid molecule containingfunctional upstream and downstream elements flanking a gene of interestwas examined using two synthetic elements containing either a T7 or a T3promoter sequence. The elements were made by annealing pairs ofsynthetic oligonucleotides. The T7 linker was generated by mixing equalmolar amounts of T7top (F9304; SEQ ID NO: 20) and T7bottom (F9305; SEQID NO: 21) oligonucleotides (FIG. 9D). The T3 linker was generated bymixing equal molar amounts of T3top (F9661; SEQ ID NO: 23) and T3bottom(F9662; SEQ ID NO: 24) oligonucleotides (FIG. 9D). The mixtures wereheated in boiling water for 5 min, then allowed to cool to roomtemperature. Both elements were designed to contain a topoisomeraserecognition site at one end.

The GFP gene was amplified with GFP primers F8418 (SEQ ID NO: 17) andF8420 (SEQ ID NO: 18, FIG. 9D; see, also, FIG. 9C). Unpurified GFP PCRproduct (2 μl) was mixed with 50 ng of T7 linker and 50 ng of T3 linker,topoisomerase was added, and the topo-joining reaction was allowed toproceed at room temperature for 5 min. Two μl of the joining reactionwas used as template for a 50 μl PCR reaction with primers for the T7and T3 sequences.

After amplification, a 4 μl aliquot of the PCR reaction was used astemplate for in vitro transcription. The reaction was performed using aPromega RiboProbe In Vitro Transcription Systems kit according to themanufacturer's instruction. The reaction was allowed to proceed for 60min at 37° C. with T7 or T3 RNA polymerase (final volume, 20 μl).Aliquots of the in vitro transcription reactions were digested withRNase or DNase, then undigested and digested samples were subjected toelectrophoresis in a 2% TBE gel. A predominant band of the predictedsize (either sense or antisense orientation) was observed in theundigested samples. No decrease in the product band was noted in samplestreated with DNase. The product bands disappeared when samples weretreated with RNase indicating the product was RNA. These resultsdemonstrate that topoisomerase can be used according to a method of theinvention to generate a ds recombinant nucleic acid molecule covalentlylinked in both strands in a predetermined orientation, and that an RNAtranscript can be expressed from such a nucleic acid molecule.

B. Expression of a Translation Product from a Topo-Ligated Construct

The ability of topoisomerase ligated polynucleotide to support coupledin vitro transcription/translation was examined. A ds recombinantnucleic acid molecule was generated according to a method of theinvention by linking an element containing a T7 promoter (plus a Kozaksequence) to lacZ PCR products of 1 kb, 2 kb, or 3 kb. Two 2 μl of thegenerated products were used as template for PCR amplification reactions(primers, SEQ ID NOS: 25-28; FIG. 9D). Unpurified aliquots of theamplification reactions (3 μl) were used as templates for coupledtranscription/translation with a TNT T7 Quick for PCR DNA Kit accordingto the manufacturer's instructions (Promega).

Two μl aliquots from each reaction were separated by electrophoresis ona Tris-glycine gel (Novex), then visualized by autoradiography, whichrevealed protein products that migrated at the expected sizes. Theseresults demonstrate that a method of the invention can be used toproduce a ds recombinant nucleic acid molecule covalently linked in bothstrands useful as a template for expressing a polypeptide by a coupledin vitro transcription/translation reaction.

C. Generation of Topo-Ligated Constructs for Performing a Two HybridAssay

Two hybrid assays provide a powerful method for detectingprotein-protein interactions in vivo. These assays are based on the factthat many eukaryotic transcriptional activators consist of twophysically and functionally separable domains, including a DNA bindingdomain, which binds to a specific DNA sequence, and a transcriptionalactivation domain, which interacts with the basal transcriptionalmachinery. The association of a transactivation domain with a DNAbinding domain can promote the assembly of a functional RNA polymeraseII complex, thereby allowing transcriptional activation, for example, ofa detectable reporter gene (Field and Song, Nature 340:245-246, 1989).Where a first protein, X, is fused to a DNA binding domain, for example,a GAL4 binding domain, and a second protein, Y, which can be the same ordifferent from X, is fused into a transactivation domain, for example, aVP16 domain, an interaction of proteins X and Y can be identified bydetecting transcription of a reporter gene having a GAL4 promoter.

The ability of a method of the invention to generate linear constructsfor expressing fusion proteins for performing a mammalian two-hybridassay was examined. PCR was used to generate GAL4 (F10779 and F12667primers; SEQ ID NOS: 1 and 3, respectively), VP16 (F10779 and F12668primers; SEQ ID NOS: 1 and 5, respectively), p53 (F12669 and F12505primers; SEQ ID NOS: 8 and 4, respectively), T antigen (F12670 andF12505 primers; SEQ ID NOS: 9 and 4, respectively), and SV40pA (F12016and F561 primers; SEQ ID NOS: 6 and 7, respectively) elements containingtopoisomerase sites at the appropriate ends. Topoisomerase was used tocreate the covalently linked, double stranded constructs GAL4+p53+SV40pAand VP16+T antigen+SV40pA, and the resultant ligation products were usedas templates for PCR amplification.

Purified GAL4+p53+SV40pA and VP16+T antigen+SV40pA PCR constructs wereco-transfected with a lacZ reporter gene (pGene/lacZ plasmid; InvitrogenCorporation, Carlsbad, Calif.) into CHO cells (6 well plate, 1×10⁵cells/well). In parallel studies, the use of plasmid vectors containingthe expression constructs was examined, as was the use of PCR reactionmixtures containing the unpurified constructs. Control reactions wereperformed using GAL4+pA and VP16+pA without inserts (negative controls)or p53+VP16 (positive control). Cells were lysed 48 hr aftertransfection and reporter gene activity was measured using abeta-galactosidase assay kit.

A high level of reporter gene activity was detected with the positivecontrol (FIG. 10, sample 3) and in the sample co-transfected with thereporter gene and the linear GAI4+p53+SV40pA and VP16+T antigen+SV40pAconstructs (FIG. 10, sample 4). Low level activity (but greater thanthat of the negative controls; samples 5, 6, 8 and 9) was detected whenthe plasmid version of the constructs was used (FIG. 10, sample 1). Lowlevel activity was also observed in the sample co-transfected with theunpurified, PCR-generated prey and bait constructs (sample 7). Theseresults demonstrate that a method of the invention can be used toprepare constructs useful for performing a two hybrid assay.

Example 3 Production and Use of Directionally Topo-Charged GatewayVectors Introduction

As a combination of Topoisomerase and GATEWAY™ recombinational cloningtechnologies, directionally Topo-charged Gateway vectors were developed.These tools facilitate easy entry into the Gateway system by alleviatingthe necessity of adding attB sites (25 base pairs) to either side of aPCR amplified ORF prior to recombination into a Donor vector. Instead, afour base tag recognition sequence (CACC) is added to the 5′ end of theORF and PCR products are then directionally TOPO-cloned to create anEntry or a Gateway compatible expression vector (See FIG. 29).

In the present Example, three Topo-Gateway vectors and one Destinationvector were created in all. Two topo entry vectors have been produced:(1) pENTR/D-TOPO® (FIG. 22), which allows ORFs directionally clonedbetween attL sites to be transferred to any of the N-terminal fusionprokaryotic and all of the eukaryotic DEST vectors; and (2)pENTR/SD/D-TOPO® (FIG. 23), which allows ORFs to be directionally topocloned downstream of a prokaryotic ribosome binding siteShine-Dalgarno). Genes cloned in this manner can be transferred toprokaryotic DEST vectors without N-terminal tags and expressed inbacteria yielding proteins with native N-termini.

One directional Topo Gateway mammalian expression vector has also beenconstructed, pcDNA/GW-DT (FIG. 19). This vector allows directionalcloning of an ORF into a pcDNA 3.1 derivative. ORFs cloned into thisvector are expressed in mammalian cells under the control of the CMVpromoter. Cloned ORFs are flanked by attB sites in the vector, allowingthem to be moved around in the Gateway system via BP and LR Clonasereactions. This vector also encodes a C-terminal V5 tag, the TK polyadenylation signal, and the neomycin (G418) resistance marker forselection of stable clones in mammalian cell lines. Finally, a GatewayDestination vector was constructed from pcDNA/GW-DT by transferring theccdB and chloramphenicol resistance cassettes.

These Topo Gateway Entry and Expression vectors improve the ease ofentry into the Gateway system by allowing the researcher to directlyclone a PCR amplified gene without the necessity of adding attB sites tothe primers and performing a BP clonase reaction.

Materials and Methods

Construction of pcDNA/GW-DT. pcDNA/GW-DT was constructed by firstreplacing the multiple cloning site in pcDNA3.1 attB (an early versionwith the BGH polyadenylation signal). This was done by digesting theparent vector with BsrGI (which cuts within each att site flanking theMCS) and inserting a double stranded oligonucleotide encoding the newMCS (FIG. 18). Once the proper insertion was confirmed, the V5/His tagand BGH polyadenylation signal were replaced with a V5 tag followed bythree stop codons (TAG, TGA, TAA) and the thymidine kinase (TK)polyadenylation signal from Herpes Simplex Virus. This was accomplishedby digesting the vector with AscI and AvrII, purification of the vectorfragment, and inserting two fragments encoding the new sequences in atriple ligation (see FIG. 19).

Construction of pcDNA-DEST 40. pcDNA-DEST 40 was created frompcDNA/GW-DT via a BP clonase reaction with pDONR221. pDONR221 wascombined with pcDNAGW-DT(sc) and BP clonase (Invitrogen Corporation;Carlsbad, Calif.) in the appropriate buffer. The reaction was incubatedaccording to the standard protocol and transformants selected for onKanamycin plates. The product, a pcDNA destination vector containingattP sites flanking the ccdB, ccdA, and chloramphenicol resistance geneswas selected on ampicillin/chloramphenicol containing media. In onealternative of this aspect of the invention, the chloramphenicolresistance gene in the cassette can be replaced by a spectinomycinresistance gene (see Hollingshead et al., Plasmid 13(1):17-30 (1985),NCBI accession no. X02340 M10241), and the Destination Vector can beselected on ampicillin/spectinomycin-containing media. It has recentlybeen found that the use of spectinomycin selection instead ofchloramphenicol selection results in an increase in the number ofcolonies obtained on selection plates, indicating that use of thespectinomycin resistance gene may lead to an increased efficiency ofcloning from that observed using cassettes containing thechloramphenicol resistance gene.

Construction of pENTR/D-TOPO™ (sc). pDONOR221 was modified by adding anadaptation sequence cassette between the attP sites by BP recombinationwith pcDNA/GW-DT(sc) creating pENTR/D-TOPO® (sc) (FIG. 22). pDONR221 wascombined with pcDNA/GW-DT (sc) and BP clonase in the appropriate buffer.The reaction was incubated according to the standard protocol exceptthat DH10BsbcC cells were used for transformation and propagation ofpENTR/D-TOPO® (sc). This cell line carries a mutation that allowsmaintenance of plasmids that carry hairpin structures (e.g. attL sites)that are in close proximity. This plasmid did not support growth of Top10 cells in selective media.

Creation of pENTR/D-TOPO® and pENTR/SD/D-TOPO®. The vectorpENTR/D-TOPO®(sc) was directionally topo charged by sequential digestionwith Not I, Asc I, and Xho I followed by ligation with the directionaltopo adapters Topo-D71, -D72, -D75 and -D76 for pENTR/SD/D-TOPO® or TopoD-73, -D74, -D75, and -D76 for pENTR/D-TOPO® overnight at 15° C. (seeFIG. 26). The adapted vectors were separated from free oligonucleotidesby isopropanol precipitation at room temperature. The purified, adaptedvector was topo charged by addition of the common annealing oligo TopoD-70, T4 Kinase, and recombinant vaccinia topoisomerase I. Afterincubation at 37° C. for 15 minutes, charged vector was purified eitherby agarose gel electrophoresis (NB JC-12, 2001-035, pg. 3) orchromatography on a 25 Q MacroPrep column (BioRad) (NB2000-0342, pg.45). Directional topo cloning efficiency was assayed by incubation of 1ng purified vector with 5 ng directional (CACC) 750 bp test insert for 5minutes at room temperature. Top 10 chemically competent cells were thentransformed with 2 μl of the cloning reaction and grown out on LB platescontaining Kanamycin as antibiotic selection.

Topo-Gateway cloning and gene expression. To test the ability of thesevectors to support Topo cloning, Gateway cloning and protein production,the gene encoding human HLA class I (accession No. D32129) was amplifiedby PCR with primers that incorporated the four base CACC tag at its 5′end immediately upstream of the ATG start codon. This PCR product wascloned into both pENTR/D-TOPO® and pENTR/SD/D-TOPO®. Ten clones fromeach HLA reaction were used in colony directional PCR reactions (d-PCR).In this study, clones were amplified with a T7 primer (binds 5′ to theattL 1 site) and 129 reverse primer (specific for the 3′ end of HLA).

In addition to the HLA gene, the gene for chloramphenicol acetyltransferase (CAT) was similarly amplified and cloned into the two entryvectors. After miniprep and digestion analysis, single clones from eachreaction were isolated and sequenced using the M13 Forward and M13Reverse primers. All entry clones were confirmed by sequencing andrecombined by L/R Clonase reaction with pcDNA/GW DEST 40 (pENTR-D-TOPO®clones) or pET DEST 42 (pENTR/SD/D-TOPO® clones). Positive clones wereconfirmed by digestion with NcoI (site appears at the 5′ end ofdirectionally adapted ORFs, caCCATGG), and NotI (data not shown). Theresulting pcDNA-DEST 40 (HLA and CAT) and pcDNA/GW-DT (HLA and CAT)constructs were then used to transfect COS cells. Cells were transfectedusing Lipofectamine 2000, 8 μg DNA and Optimem buffer. Reactions wereapplied to the cells for 5 hours then the media changed. After anovernight incubation at 37° C., the cells were harvested, lysed and runon a 4-20% Tris-Glycine gel using standard procedures. Afterelectrophoresis, proteins were transferred to nitrocellulose membranes,blocked, and probed with V5-HRP antibody and ECL detection.

One positive clone from each pET DEST 42 reaction was used to transformBL21(DE3) cells and grown overnight in LB/Amp. The culture was thendiluted 1:25 in the same medium and allowed to grow to O.D. (600 nm)=0.5at which time expression of recombinant protein was induced by additionof IPTG to a final concentration of 1 mM. After the cultures wereallowed to grow 3 hours at 37° C., cells were harvested bycentrifugation. Aliquots of cell pellets were boiled in NuPagedenaturing sample buffer, run on 4-12% NuPage polyacrylamide gels, andstained using SafeStain™ (Invitrogen Corporation, Carlsbad, Calif.). Asa positive control for expression of test genes in the pET DEST 42vector, the HLA and CAT genes were directly topo cloned into pET100 CATand HLA (dTopo, no attB sites). These constructs were used to transfectBL21(DE3) E. coli cells, grown to log phase and induced with IPTG asdescribed above.

Results and Discussion

Directional cloning efficiency of HLA and CAT clones in pENTR-dTopo andpENTR/SD-dTopo. Directional PCR reactions were designed to ensure thatthe HLA ORF cloned into pENTR/D-TOPO®″ and pENTR/SD/D-TOPO® were in thecorrect orientation. Ten colonies were picked from each of the Topocloning transformations and put directly into PCR reactions as describedin “Materials and Methods.” Eight of ten pENTR/SD-HLA clones tested werecorrectly oriented while nine of ten pENTR-HLA clones were correct.These tests were done with gel purified vector which had approximately10-15% no insert background (data not shown).

Alternatively, restriction analysis of the CAT clones was done. Cloneswere isolated and the DNA digested with NcoI and AscI. One of the twoNcoI sites in a correctly oriented CAT clone appears at the 5′ end ofeach ORF as part of the Kozac directional adaptation sequence and thefirst two codons of the CAT gene (caCCATGG). AscI is present in thevector at the 3′ end of the ORF. A correctly oriented clone will havetwo NcoI sites (one at the 5′ end and one internal) and will yield 500bp and 150 bp fragments after a double digest with Asc I. The CAT ORFencodes at its 3′ end the sequence, CGCC, which is a one base pairmismatch to the optimum tag sequence. This close homology caused the CATPCR product to directionally clone with only 50% efficiency (four ofeight clones, data not shown).

Sequencing of Entry Clones. Each of the Entry clones chosen forrecombination into DEST vectors and subsequent expression were sequencedfrom both ends to confirm that the adapters and ORFs ligated correctly.M13 forward and reverse primers were used and the reactions were sent toResGen for sequencing on an ABI 3700 capillary sequencer. From thesereactions a minimum of 600 bases of readable sequence were obtained. Itis clear that there is some loss of signal as the reaction proceedsthrough the attL sites but significant signal remains after this pointusing this procedure (data not shown).

Expression of HLA and CAT in COS cells. Expression from pcDNA/GW/D-TOPO®and pcDNA-DEST 40 was tested by transfection of COS cells with HLA andCAT as the test gene in these constructs. Harvested lysates were probedfor V5-tagged recombinant protein by Western blot using the V5 antibody.Data shown in FIG. 27 indicates that both the HLA and CAT genes expressin these vectors whether the genes were cloned directly via Topo cloning(FIG. 27, lanes 3 and 6) or after LR clonase transfer from pENTR/D-TOPO®(FIG. 27, lanes 2 and 5).

Bacterial expression of HLA and CAT. The CAT and HLA genes cloned intopENTR/SD/D-TOPO® were transferred via LR Clonase reaction to pDEST-42(pET, C-terminal V5/His). The results shown in FIG. 28 suggest that theCAT gene expressed in bacteria whether it is flanked by attB sites ornot (FIG. 28, compare lanes 6 and 7). The finding that the CAT geneexpresses well in E. coli after being transferred to a pET DEST vectorfrom pENTR/SD/D-TOPO® validates the utility of this system for cloningand expressing ORFs using the Topo-Gateway system.

Interestingly, HLA cloned into pDEST 42 (flanked by attB sites) failedto express in BL21(DE3) cells in two independent studies (FIG. 28, lanes3 and 4). As seen above, the HLA gene from the same Entry cloneexpressed well in COS cells when recombined into a mammalian DESTvector. Further, the fact that the pET system was unable to supportexpression of the HLA gene when it was flanked by attB sites suggeststhat there can be gene specific variations on expression using theGateway system at least in bacteria. One factor that may be involved inthis result is that HLA expressed from the control vector (pET 100d-Topo) ran anomalously in the gel (30 kDa instead of the predicted 41kDa). This human protein may not express well in bacteria in any caseand the expression problem may be exacerbated by addition of attB sites.

In conclusion, we have described the construction and testing of two newTopo Gateway Entry vectors, one new Topo Gateway Expression vector and anew DEST vector that followed from that. In all, these new tools thatcombine the ease and efficiency of Topo cloning and the versatility ofthe Gateway system permit the cloning and expression of large numbers ofgenes in many different contexts with a minimum of expense and effort.

Example 4 Alternative Methods of Topoisomerase Cloning

In one preferred alternative embodiment of the present invention, a TOPOSSS vector is made by first obtaining a commercially available cloningvector. One such vector is pUni/V5-His version A (InvitrogenCorporation, Carlsbad, Calif.), a circular supercoiled vector thatcontains uniquely designed elements. These elements include a BGHpolyadenylation sequence to increase mRNA stability in eukaryotic hosts,a T7 transcription termination region, an R6Kg DNA replication originand a kanamycin resistance gene and promoter for antibiotic resistanceselection. Additionally, pUni/V5-His version A contains a multiplecloning site, which is a synthetic DNA sequence encoding a series ofrestriction endonuclease recognition sites. These sites are engineeredfor cloning of DNA into a vector at a specific position. Also within thevector's multiple cloning site is a loxP site inserted 5′ to theendonuclease recognition sites thereby facilitating Crerecombinase-mediated fusion into a variety of other expression vectors,(Echo™ Cloning System, Invitrogen Corporation, Carlsbad, Calif.). Anoptional C-terminal V5 epitope tag is present for easy detection ofexpressed fusion proteins using an Anti-V5 Antibody. An optionalC-terminus polyhistidine (6×His) tag is also present to enable rapidpurification and detection of expressed proteins. A bacterial ribosomalbinding site downstream from the loxP site makes transcriptioninitiation in E. coli possible. Though this combination of elements isspecific for pUni/V5-His version A cloning vector, many similar cloningand expression vectors are commercially available or may be assembledfrom sequences and by methods well known in the art. pUni/V5-His versionA is a 2.2 kb double stranded plasmid.

Construction of a topoisomerase I charged cloning vector frompUni/V5-His version A is accomplished by endonuclease digestion of thevector, followed by complementary annealing of syntheticoligonucleotides and site-specific cleavage of the heteroduplex byVaccinia topoisomerase I. SacI and EcoRI are two of the many restrictionendonuclease sites present within the multiple cloning site ofpUni/V5-His version A. Digestion of pUni/V5-His version A with thecorresponding restriction enzymes, SacI and EcoRI will leave cohesiveends on the vector (5′-AGCT-3′ and 5′-AATT-3′). These enzymes arereadily available from numerous vendors including New England Biolabs(Beverly, Mass., Catalogue Nos. R0156S, SacI and RO101S, EcoRI). Thedigested pUni/V5-His version A is easily separated from the digestedfragments using isopropanol precipitation. These and other methods fordigesting and isolating DNA are well known to those of ordinary skill inthe art (Sambrook, J., Fritsch, E. F., and T. Maniatis. (1989) MolecularCloning, A Laboratory Manual. Second edition. Cold Spring HarborLaboratory Press. pp 5.28-5.32.)

The purified, digested vector is then incubated with two specificoligonucleotide adapters and T4 DNA ligase. The adapters areoligonucleotide duplexes containing ends that are compatible with theSacI and EcoRI ends of the vector. One of skill in the art will readilyappreciate that other adapter oligonucleotides with appropriatesequences can be made for other vectors having different restrictionsites. Following incubation with T4 DNA ligase, the vector containingthe ligated adapters is purified using isopropanol. The adapter duplexthat results from the annealing of TOPO D1 and TOPO D2 has asingle-stranded Eco R1 overhang at one end and a 12-nucleotidesingle-stranded overhang at the other end.

The first adapter oligonucleotide, (TOPO D1), has complementation to theEcoRI cohesive end, 3′-TTAA-5′. Furthermore, TOPO D1 has an additional24-bp including the topoisomerase consensus pentapyriridine element5′-CCCTT located 16-bp upstream of the 3′ end. The remaining sequenceand size of TOPO D1 adapter oligo is variable, and may be modified tofit a researcher's particular needs. According to one such aspect ofthis preferred embodiment of the invention,

5′-AATTGATCCCTTCACCGACATAGTACAG-3 (SEQ ID NO: 33)is the full sequence of the adapter used.

The second adapter oligonucleotide, (TOPO D2), must have fullcomplementation to TOPO. D1. TOPO D2 complements directly 5′ of theEcoRI cohesive flap, extending the bottom strand of the linearizedvector. Additionally, TOPO D2 contains the sequence 3′-GTGG, which isthe necessary SSS for directional cloning. In this embodiment, the SSSwas chosen to complement the Kozak sequence known to help expression ofORFs in eukaryotic cells by increasing the efficiency of ribosomebinding on the mRNA, however, sequence and length are highly variable tomeet the specific needs of individual users. The complete sequence ofTOPO D2 is 3-CTAGGGAAGTGG-5 (SEQ ID NO:34). Similar to above, theadapter duplex that results from the annealing of oligonucleotides TOPOD4 and TOPO D5 has a single-stranded SacI overhang at one end, and a 12nucleotide single-stranded overhang at the other end.

The third adapter oligonucleotide (TOPO D5), has complementation to theSacI cohesive end, 3′-TCGA-5′. Similar to TOPO D1, TOPO D5 hasadditional bases creating a single stranded overhang. The length andsequence can vary based on the needs of the user. In the currentembodiment TOPO D5's sequence is 5′-AAGGGCGAGCT-3′ (SEQ ID NO:35).

The fourth adapter oligonucleotide (TOPO D4), has full complementationto TOPO D5, and complements directly 5′ of the SacI cohesive flapextending the top strand of the linearized vector. TOPO D4 also containsthe topoisomerase consensus sequence 5′-CCCTT. The remaining sequenceand size of TOPO D4 adapter oligo is variable and may be modified to fitparticular needs. In the current embodiment, the sequence of TOPO D4 is3′-GACATGATACAGTTCCCGC-5′ (SEQ ID NO:36), which includes an additional12 bp single stranded overhang.

These adapter oligonucleotides can be chemically synthesized using anyof numerous techniques, including the phosphoramadite method,(Caruthers, M. H., Barone, A. D., Beaucage, S. L., Dodds, D. R., Fisher,E. F., McBride, L. J., Matteucci, M., Stabinsky, Z., and Tang, J. Y.,(1987) Chemical Synthesis of Deoxyoligonucleotides, Methods Enzymol.154: 287-313). This and other methods for the chemical synthesis ofoligos are well known to those of ordinary skill in the art.

Complementary annealing of the purified digested vector and the adapteroligonucleotides is done by incubation of the DNA in the presence of T4DNA ligase. Typical ligation reactions are performed by incubation of acloning vector with suitable DNA fragments in the presence of ligase andan appropriate reaction buffer. Buffers for ligation reactions shouldcontain ATP to provide energy to for the reaction, as well as, reducingreagents like dithiothreitol and pH stabilizers like Tris-HCl. The ratioof concentrations for the cloning vector and the DNA fragments aredependent on each individual reaction, and formulae for theirdetermination are abundant in the literature, (See e.g. Protocols andApplications Guide (1991), Promega Corporation, Madison, Wis., p. 45).T4 Ligase will catalyze the formation of a phosphodiester bond betweenadjacent 5′-phosphates and 3′-hydroxyl termini during the incubation.Cohesive end ligation can generally be accomplished in 30 minutes at12-15° C., while blunt end ligation requires 4-16 hours at roomtemperature, (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,Seidman, J. G., Smith, J. A., Struhl, K. (1992) Second Edition; ShortProtocols in Molecular Biology, John Wiley & Sons, Inc., New York, N.Y.,pp. 3.14-3.37), however parameter range varies for each study. In thecurrent embodiment, purified, digested pUni/V5-His version A and theadapter oligos were incubated in the presence of T4 ligase and asuitable buffer for sixteen hours at 12.5° C. The resulting linearizedand adapted vector comprises the purified cloning vector attached to theadapter oligonucleotides through base pair complementation and T4ligase-catalyzed, phosphodiester bonds.

Efficient modification of the adapted vector with topoisomerase requiresthe addition of an annealing oligo to generate double, stranded DNA onTOPO D1's and TOPO D4's single stranded overhangs. Vacciniatopoisomerase I initially binds noncovalently to double stranded DNA.The enzyme then diffuses along the duplex until locating and covalentlyattaching to the consensus pentapyrimidine sequence 5′-CCCTT, formingthe topoisomerase adapted complex (See Shuman et al., U.S. Pat. No.5,766,891). Modification of the adapted vector takes place in theabsence of DNA ligase to prevent the formation of phosphodiester bondsbetween the adapted vector and the annealing oligo, since phosphodiesterbonds in the non-scissile strand will prevent the dissociation of theleaving group upon cleavage.

The annealing oligonucleotide (TOPO D3), must have complementation tothe single stranded DNA overhangs of TOPO D1 and TOPO D4. In the currentembodiment the overhangs both share the following sequence,5′-GACATAGTACAG-3′ (SEQ ID NO:37). Therefore, TOPO D3 has the followingsequence, 3-CTGTATCATGTCAAC-5 (SEQ ID NO:38), which comprises fullcomplementation to the adapter oligos' single stranded overhang and anadditional 3 bp overhang, 3′-AAC-S′.

Incubation of the adapted vector with the annealing oligo in thepresence of topoisomerase will create double stranded DNA to whichtopoisomerase can noncovalently bind. Bound topoisomerase will searchthe double stranded DNA by a facilitated diffusion mechanism, until the5′-CCCTT recognition motif is located. Cleavage of the phosphodiesterbackbone of the scissile strand 3′ of the motif is catalyzed via anucleophilic attack on the 3′ phosphorus atom of the preferredoligonucleotide cleavage sequence 5-CCCTT, resulting in covalentattachment of the DNA to the enzyme by a 3′-phosphotyrosyl linkage, (SeeShuman, S., Kane, E. M., Morham, S. G. (1989) Proc. Natl. Acad. Sci.U.S.A. 86, 9793-9796). Cleavage of the scissile strand creates a doublestranded leaving group comprising the 3′ end adapter oligo, downstreamfrom the 5′-CCCTT motif, and the annealing oligo TOPO D3. Although theleaving group can religate to the topoisomerase-modified end of thevector via 5′ hydroxyl-mediated attack of the phosphotyrosyl linkage,this reaction is disfavored when the leaving group is no longercovalently attached to the vector. The addition of T4 polynucleotidekinase and ATP to the cleavage/religation reaction further shifts theequilibrium toward the accumulation of trapped topoisomerase since thekinase can phosphorylate the 5′ hydroxyl of the leaving group to preventthe rejoining from taking place, (Ausubel, F. M., Brent, R., Kingston,R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. (1992)Second Edition; Short Protocols in Molecular Biology, John Wiley & Sons,Inc., New York, N.Y., pp. 3.14-3.30). The resulting linearized vectorcomprises a blunt end from the TOPO D4/D3 leaving group and a SSSbearing end from the TOPO D1/D3 leaving group. Both of the linearizedcloning vector's ends are charged with topoisomerase, enabling fast,efficient and directional topoisomerase mediated insertion of anacceptor molecule.

Although the above example details the modification of pUni/V5-Hisversion A to form the topoisomerase-modified directional cloning vector,a person of ordinary skill in the art will appreciate how to apply thesemethods to any plasmid, cosmid, virus, or other DNA. It should also benoted that this example demonstrates a vector containing a 5′single-stranded overhang comprising the sequence 5′-GGTG-3′, however thedesign of adapter duplexes and annealing oligonucleotides would allowone of skill in the art to custom design overhangs of any sequence orlength at one or both ends of a given vector.

Specifically, any plasmid, cosmid, virus or other DNA can be modified topossess a SSS of any convenient sequence and length. These are the basicsteps: the vector is first subjected to a treatment that is known tolinearize the DNA. Common procedures include, but are not limited to,restriction digestion and treatment with topoisomerase II. Followinglinearization, a custom SSS is added. In the above example,complementary oligonucleotides are added to the sticky ends of arestriction digestion giving the desired SSS, however SSS formingoligonucleotides can be added by T4 blunt end ligation, as well. The SSSsequence is exposed by a topoisomerase I mediated, single strandnicking. In turn, this SSS can be used to directionally insert a PCRproduct comprising one or more complimentary SSS.

Likewise, topoisomerase modification can be applied to anydouble-stranded plasmid, cosmid, virus or other piece of DNA. Methodsfor the attachment of topoisomerase I to double stranded DNA are wellknown in the art, (See Shuman et al., U.S. Pat. No. 5,766,891). Thestrategic placement of topoisomerase on to a piece of double strandedDNA is determined by the incorporation of a topoisomerase I consensussequence, (See Shuman et al., U.S. Pat. No. 5,766,891). Thetopoisomerase I will bind the double stranded DNA, nick the scissilestrand thus revealing the predetermined single-stranded overhangsequence, and ligate the incoming PCR product in the correct, SSSmediated orientation.

Example 5 Production of Custom Topoisomerase I-Adapted Vectors

As an example of the application of this aspect of the current inventionto another plasmid, pCR 2.1 (Invitrogen Corporation; Carlsbad, Calif.)was modified to create a topoisomerase I adapted vector with a customsingle stranded sequence.

Plasmid pCR 2.1 is 3.9 kb T/A cloning vector. Within the sequence ofthis vector are many uniquely designed elements. These elements includean f1 origin, a ColE1 origin, a kanamycin resistance gene, an ampicillinresistance gene, a LacZ-alpha fragment and a multiple cloning sequencelocated within the LacZ-alpha fragment allowing for blue-white selectionof recombinant plasmids. The multiple cloning sequence of pCR 2.1contains; numerous restriction sites, including but not limited to,HindIII, SpeI and EcoRI; M13 forward and reverse primers and a T7 RNApolymerase promoter.

Construction of the topoisomerase I charged vector possessing a customsingle stranded sequence consists of endonuclease digestion followed bycomplementary annealing of synthetic oligonucleotides and the sitespecific cleavage of the heteroduplex by Vaccinia topoisomerase I.Digestion of pCR 2.1 with the restriction enzymes HindIII, SpeI andEcoRI leaves HindIII and EcoRI cohesive ends on the vector. Thedissociated fragment of pCR 2.1 downstream from the HindIII cleavagesite is further cleaved with SpeI in order to reduce its size. Byreducing the size of the fragment, the digested vector is easilypurified away from the smaller digested pieces by isopropanolprecipitation. These enzymes are readily available from numerous vendorsincluding New England Biolabs, (Beverly, Mass., Catalogue Nos.; RO104S,HindIII; RO133S, SpeI; RO101S, EcoRI). Methods for the digestion and theisolation of DNA are well known to those skilled in the art, (Sambrook,J., Fritsch, E. F., and T. Maniatis. (1989) Molecular Cloning, ALaboratory Manual. Second edition. Cold Spring Harbor Laboratory Press.pp. 5.28-5.32.)

The purified digested vector is incubated with four adapteroligonucleotides and T4 DNA ligase. These adapter oligonucleotides aredesigned to have complementation to either the HindIII cohesive end, theEcoRI cohesive end, or to each other. Following incubation with T4 DNAligase the adapted vector is purified using isopropanol.

The first adapter oligonucleotide, (TOPO H), has complementation to theHindIII cohesive end, 3′-TCGA-5′. Furthermore, TOPO H has an additional24 bp including the topoisomerase consensus pentapyrimidine element5′-CCCTT located 19-bp upstream of the 3′ end. The remaining sequenceand size of TOPO H adapter oligo is variable, and may be modified to fita researcher's particular needs. In the current embodiment

5′-AGCTCGCCCTTATTCCGATAGTG-3′ (SEQ ID NO: 39)is the full sequence of the adapter used.

The second adapter oligonucleotide (TOPO 16), must have fullcomplementation to TOPO H. TOPO 16 complements directly 5′ of theHindIII cohesive end, extending the bottom strand of the linearizedvector. Additionally, TOPO 16 contains the sequence 3′-TAAG, which isthe chosen single stranded sequence for directional cloning. Thecomplete sequence of TOPO 16 is 3′-GCGGGAATAAG-5′ (SEQ ID NO:40).

The third adapter oligonucleotide (TOPO 1), has complementation to theEcoRI cohesive end, 3′-TTAA-5′. Similar to TOPO H, TOPO 1 has additionalbases containing the topoisomerase I consensus sequence CCCTT located 12bp upstream of the 3′ end. The length and sequence of TOPO 1 can varybased on the needs of the user. In the current embodiment TOPO 1'ssequence is 5′-AATTCGCCCTTATTCCGATAGTG-3′ (SEQ ID NO:41).

The fourth adapter oligonucleotide (TOPO 2), has full complementation toTOPO 1, and complements directly 5′ of the EcoRI cohesive end extendingthe top strand of the linearized vector. In the current embodiment, thesequence of TOPO 2 is 3′-GCGGGAA-5′.

Complementary annealing of the purified digested vector and the adapteroligonucleotides is done by incubation of the DNA in the presence of T4DNA ligase. T4 Ligase will catalyze the formation of a phosphodiesterbond between adjacent 5′-phosphates and 3′-hydroxyl termini during theincubation. In the current embodiment, purified, digested pCR 2.1 andthe adapter oligos were incubated in the presence of T4 ligase and asuitable buffer for sixteen hours at 12.5° C. The resulting linearizedand adapted vector comprises the purified cloning vector attached to theadapter oligonucleotides through base pair complementation and T4ligase-catalyzed, phosphodiester bonds. Ligation techniques are abundantin the literature, (see Ausubel, F. M., et al, (1992) Second Edition;Short Protocols in Molecular Biology, John Wiley & Sons, Inc., New York,N.Y., pp. 3.14-3.37)

Charging of the adapted vector with topoisomerase requires the additionof annealing oligonucleotides to generate double stranded DNA on TOPOH's and TOPO1's single stranded overhangs. Charging of the adaptedvector takes place in the absence of DNA ligase to prevent the formationof phosphodiester bonds between the adapted vector and the annealingoligo, since phosphodiester bonds in the non-scissile strand willprevent the dissociation of the leaving group upon cleavage.

The annealing oligonucleotide (TOPO 17), must have complementation tothe single stranded DNA overhang of TOPO H. In the current embodimentthe overhang has the following sequence, 5′-CGATAGTG 3′. Therefore, TOPO17 has the following sequence, 3′-GCTATCAC 5′, which comprises fullcomplementation to the adapter oligo's single stranded overhang.

The annealing oligonucleotide (TOPO 3), must have complementation to thesingle stranded DNA overhang of TOPO 1. In the current embodiment theoverhang has the following sequence, 3′-GTGATAGCCTTA-5′ (SEQ ID NO:42).Therefore, TOPO 3 has the following sequence, 5′-CAACACTATCGGAAT-3′ (SEQID NO:43), which comprises full complementation to the adapter oligo'ssingle stranded overhang and an additional 3 bp overhang, 5′-CAA-3′.

Incubation of the adapted vector with the annealing oligo in thepresence of topoisomerase will create double stranded DNA to whichtopoisomerase can noncovalently bind. Bound topoisomerase will searchthe double stranded DNA by a facilitated diffusion mechanism, until the5′-CCCTT recognition motif is located. Cleavage of the phosphodiesterbackbone of the scissile strand 3′ of the motif will result in thecovalent attachment of the DNA to the enzyme by a 3′-phosphotyrosyllinkage, (See Shuman, S., et al (1989) Proc. Natl. Acad. Sci. U.S.A. 86,9793-9796). Cleavage of the scissile strand creates a double strandedleaving group comprising the 3′ end the adapter oligos, downstream fromthe 5′-CCCTT motif, and the complementary annealing oligonucleotide. Theleaving group can religate to the topoisomerase adapted vector throughits 5′ hydroxyl's attack of the phosphotyrosyl linkage, also catalyzedby topoisomerase. Addition of T4 polynucleotide kinase to theequilibrium reaction prevents the back reaction via the kinase-mediatedphosphorylation of the leaving group's 5′ hydroxyl, (Ausubel, F. M., etal (1992) Second Edition; Short Protocols in Molecular Biology, JohnWiley & Sons, Inc., New York, N.Y., pp. 3.14-3.30). The resultinglinearized vector comprises a blunt end from the TOPO 1/3 leaving groupand a single stranded sequence end from the TOPO H/17 leaving group.Both of the linearized cloning vector's ends are charged withtopoisomerase, enabling fast, efficient and directional topoisomerasemediated insertion of an acceptor molecule.

Example 6 Directional Cloning Using Topoisomerase

This aspect of the invention also provides a method for directionalcloning of DNA. In such methods, the TOPO SSS vector constructed frompUni/V5-His version A was used for the directional insertion of ORFsfrom the GeneStorm Expression Ready Clones (Invitrogen Corporation,Carlsbad, Calif.). The modified pUni vector was selected for the cloningof these ORF's because the single strand added to the vector hashomology to the Kozak sequence known to enhance ORF expression. Note,however, that, as before, any plasmid, cosmid, virus or other DNA couldbe modified to possess the necessary single stranded sequence. Likewise,any DNA fragment could be modified to possess a homologous sequence toany vector SSS. As a point of interest, the sequence of the SSS caneffect directional cloning efficiencies. For example, SSSs with low GCcontent will have lower annealing stability, also SSSs that have highcomplementation to both ends of a DNA fragment to be cloned will loosethe capability to direct these DNA inserts. Thus the sequence of a SSSshould be carefully designed to avoid these and similar problems.

This aspect of the present invention is particularly useful in thedirectional insertion of PCR products into vectors constructed accordingto the present invention. In the PCR amplification of the desiredinsert, the PCR primers are designed so as to complement identifiedsequences of the insert(s) that are to be directionally cloned into theTOPO SSS vector. The primer designed to bind upstream of the DNA'scoding strand is modified with an additional vector SSS complementationsequence on its 5′ end. The resulting PCR product will possess acomplementary sequence allowing SSS mediated directional insertion intothe TOPO SSS cloning vector and subsequent expression of the product.

One such embodiment comprises introducing to a donor duplex DNAsubstrate a SSS site by PCR amplifying the donor duplex DNA moleculewith the 5′ oligonucleotide primer containing the SSS. PCR amplificationof a region of DNA is achieved by designing oligonucleotide primers thatcomplement a known area outside of the desired region. In a preferredembodiment the primer that has homology to the coding strand of thedouble stranded region of DNA will possess an additional sequence ofnucleotides complementary to the SSS of the TOPO SSS cloning vector.

Using the current invention in a high throughput format, we selected 82known ORFs from the GeneStorm expression system (Invitrogen Corporation,Carlsbad, Calif.) for directional cloning into the TOPO SSS vector,however, any sequence of DNA may be selected as desired by individualusers. For each of these ORFs, primers are designed with homology to thecoding and the non-coding strands. To clone PCR products in adirectional fashion into the modified pUni/V5-His version A TOPO SSSvector as described in Example 4, one primer of a given pair wasmodified to contain the nucleotide sequence complementary to the SSScontained within the vector. In the current example, the coding primercontained the added sequence 5′-CACC-3′, which complements the ‘SSS’,3′-GTGG-5′, of the TOPO SSS cloning vector. PCR amplification of theabove ORFs with their respective primers will produce double strandedDNA fragments, which possess the SSS at their 5′ end. We used Pfupolymerase in our PCR amplification, but it is well-known that PCRreactions can be performed with either a non-thermophillic polymerasesuch as Pfu or with a thermophillic polymerase like Taq followed by ablunting step to remove the non-template nucleotide these enzymes leaveat the end of PCR products.

In the present example, 0.1 μg of each primer was combined with 0.05 μgof DNA containing an ORF in a PCR reaction mix totaling 50 μl totalvolume. Besides the primers and vector, the reaction mix also containedwater, PCR buffer salts, 10 mM dNTPs and 1.25 units of Pfu polymerase.Thermal cycling temperatures were as follows: an initial 94° C.denaturation; followed by 25 repetitions of 94° C. denaturation, 55° C.primer annealing, and 72° C. elongation, each at one minute; and endedwith a 72° C., fifteen minute elongation. These parameters will varywith each DNA fragment to be amplified, and can be optimized forfragments of varying lengths and composition using methods well known tothose of ordinary skill in the art (Ausubel, F. M., Brent, R., Kingston,R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. (1992)Second Edition; Short Protocols in Molecular Biology, John Wiley & Sons,Inc., New York, N.Y., pp. 15.3-15.4). Techniques for the conversion of3′ overhangs to blunt end termini will also be familiar to those ofordinary skill in the art (Protocols and Applications Guide (1991),Promega Corporation, Madison, Wis., pp. 43-44).

Incubation of the PCR amplified donor duplex DNA containing the SSScomplementary sequence with the modified pUni/V5-His version A TOPO SSSvector results in the directional cloning of the donor DNA. For example,the eighty-two ORFs from the GeneStorm clone collection (InvitrogenCorporation, Carlsbad, Calif.) were amplified using SSS adapted primers.Amplification of the 82 GeneStorm ORFs with the described modifiedprimer pairs resulted in PCR products that had the SSS complementarysequence at their 5′ end. This ORF PCR product is combined with 10 ng ofTOPO SSS cloning vector in either sterile water or a salt solution. Thereaction is mixed gently and incubated for 5 minutes at room temperature(22-23° C.). After five minutes, we placed the reaction on ice thenproceeded to the OneShot® Chemical Transformation or Electroporation(Invitrogen Corporation, Carlsbad, Calif., Catalogue Nos. C4040-10 andC4040-50, respectively) (Invitrogen TOPO Cloning Protocol. InvitrogenCorporation Carlsbad, Calif.). Topoisomerase had joined the adjacentstrands of the vector and the product by catalyzing a rejoining reaction(FIG. 29). DNA fragments constructed with the SSS at their 5′ ends werethus correctly inserted into TOPO SSS cloning vectors with a highefficiency.

Directional insertion of DNA fragments containing 5′ SSS occurs withgreater than 90% efficiency as shown by sequencing multiple colonies oftransformed host cells. In the current example, the TOPO SSS cloningvectors containing the GeneStorm ORFs were incubated with transformationcompetent E. coli host cells. In 74 of the transformation reactions, thedirectional cloning of the ORFs into the TOPO SSS cloning vectoroccurred in at least seven of the eight colonies picked, and 59 of thesecloning reactions were directional in all eight colonies picked. Theoverall directional cloning score was 609 of 656, thus, directionalinsertion was present in over 93% of the clones picked (see Table 5).

TABLE 5 Directional Cloning of ORFs using a TOPO SSS Cloning VectorPositive colonies. dPCR reactions Clones tested 8/8 59 7/8 15 6/8 2 5/81 4/8 3 3/8 2

Example 7 Directional Cloning of a Reporter Gene

In a similar example, using the above described modified pCR2.1 TOPO SSSvector, a PCR-generated ORF encoding the gene encoding the reportermolecule Green Fluorescent Protein (GFP) was directionally cloned inframe with the lacZ a fragment present in the vector. The primers usedto amplify the GFP gene contained the requisite SSS complementationsequence 5′-ATTC-3′, and the known sequence for translation initiatingmethionine, 5′-ATG-3′. Using the necessary cloning steps noted above,the PCR amplified GFP was inserted into the vector and transformed cellswere grown on solid Agar plates. Glowing colonies represented acorrectly inserted PCR product (see Table 6).

TABLE 6 In-frame and Directional Insertion of GFP Into Modified pCR2.1TOPO SSS Cloning Vector. 5′ sequence of PCR Percentage of Correct TotalWhite Product Inserts Colonies 5′-ATTCATG-3′ 86% 457 (homologous)5′-CAAGATG-3′ 35% 118 (non-homologous) 5′-ATTCGGATG-3′ 0% 268 (frameshift) VECTOR ONLY 0% 31

These data represent a substantial improvement over the current state ofthe art in cloning, and furthermore present an invention in cloning thatis highly compatible with high throughput techniques. Given directionalcloning efficiencies greater than 90%, a user need only screen twocolonies for each cloned DNA fragment. Thus, on a 96-well plate, 48separate clones can be screened for directional insertion, 400% morethan current cloning techniques. Use of this invention will streamlinemany high-throughput-gene-expression operations, and allow them to berun at a fraction of their current costs.

Example 8 Directional Topoisomerase Cloning of Blunt-End PCR Productsinto Entry Vectors Overview

In additional embodiments, the compositions, kits and methods of theinvention combine a highly efficient, 5-minute cloning strategy (“TOPO®Cloning;” Invitrogen Corporation, Carlsbad, Calif.) to directionallyclone blunt-end PCR products into vectors for entry into therecombinational cloning system of the invention (e.g., the GATEWAY™System available from Invitrogen Corporation, Carlsbad, Calif.). Usingthis cloning strategy of the invention, blunt-end PCR products clonedirectionally at greater than 90% efficiency, with no ligase, post-PCRprocedures, or restriction enzymes required.

For optimal expression of a PCR product after recombination with theGATEWAY™ destination vector of interest, any suitable expression vectormay be used. Examples include, but are not limited to, the pENTRDirectional TOPO® vectors available commercially (InvitrogenCorporation; Carlsbad, Calif.), which have a number of benefitsincluding the following:

Vector Benefits

-   -   pENTR/D-TOPO® For efficient expression of a gene of interest        after recombination with a GATEWAY™ destination vector        pENTR/SD/D-TOPO® Contains a T7 gene 10 translational enhancer        and a ribosome binding site for optimal expression of native        protein after recombination with a prokaryotic GATEWAY™        destination vector        -   Also suitable for efficient expression of a gene of interest            in other host cell systems (e.g., mammalian, insect, yeast)            after recombination with a suitable GATEWAY™ destination            vector

These pENTR/D-TOPO® and pENTR/SD/D-TOPO® vectors are designed tofacilitate rapid, directional TOPO® Cloning of blunt-end PCR productsfor entry into the GATEWAY™ System. Features of these vectors include:

-   -   attL1 and attL2 sites for site-specific recombination of the        entry clone with a GATEWAY™ destination vector;    -   Directional TOPO® Cloning site for rapid and efficient        directional cloning of blunt-end PCR products;    -   rmB transcription termination sequences to prevent basal        expression of the PCR product of interest in E coli;    -   Kanamycin resistance gene for selection in E. coli;    -   pUC origin for high-copy replication and maintenance of the        plasmid in E. coli; and    -   T7 gene 10 translation enhancer and ribosome binding site for        efficient translation of the PCR product in prokaryotic systems        (pENTR/SD/D-TOPO® only).

Using these pENTR Directional TOPO® vectors in conjunction with theGATEWAY™ recombinational cloning system of the invention, genes ofinterest contained in blunt-end PCR products may be readily expressed byfollowing several simple steps:

1. the blunt-end PCR product is cloned (using topoisomerase in the“TOPO® Cloning” procedures described herein) into one of the pENTR TOPO®vectors described above, to generate an entry clone;

2. an expression construct is generated by performing a recombinationreaction between this entry clone and a GATEWAY™ destination vector ofchoice (such as those described elsewhere herein); and

3. the expression construct is introduced into an appropriate host cell(e.g., a bacterial, mammalian, yeast, insect, or other appropriate hostcell, the choice depending on the specific destination vector chosen forproduction of the expression construct above), and the recombinantprotein encoded by the gene of interest on the PCR product (and nowcontained on the expression construct) is expressed using expressionconditions appropriate for the particular host cell system.

Directional TOPO® Cloning

Topoisomerase I from Vaccinia virus binds to duplex DNA at specificsites (CCCTT) and cleaves the phosphodiester backbone in one strand(Shuman, 1991). The energy from the broken phosphodiester backbone isconserved by formation of a covalent bond between the 3′ phosphate ofthe cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I.The phospho-tyrosyl bond between the DNA and enzyme can subsequently beattacked by the 5′ hydroxyl of the original cleaved strand, reversingthe reaction and releasing topoisomerase (Shuman, 1994). TOPO® Cloningexploits this reaction to efficiently clone PCR products.

Directional joining of double-stranded DNA using TOPO®-chargedoligonucleotides occurs by adding a 3′ single-stranded end (overhang) tothe incoming DNA (Cheng and Shuman, 2000). This single-stranded overhangis identical to the 5′ end of the TOPO®-charged DNA fragment. By thepresent invention, this approach has been modified by adding a 4nucleotide overhang sequence to the TOPO®-charged DNA and adapting it toa “whole vector” format.

In this system, PCR products are directionally cloned by adding fourbases to the forward primer (CACC). The overhang in the cloning vector(GTGG) invades the 5′ end of the PCR product, anneals to the addedbases, and stabilizes the PCR product in the correct orientation.Inserts can be cloned in the correct orientation with efficiencies equalto or greater than 90%.

Methods

Designing PCR Primers. The design of the PCR primers to amplify a geneof interest is critical for expression. Depending on the pENTR TOPO®vector being used, several considerations must be kept in mind duringdesign of PCR primers, including:

-   -   the sequences required to facilitate directional cloning;    -   the sequences required for proper translation initiation of the        PCR product; and    -   whether or not the PCR product is to be fused in frame with an        N- or C-terminal tag after recombination of the entry clone with        a GATEWAY™ destination vector.

Guidelines to Design the Forward PCR Primer. When designing the forwardPCR primer, the following points must be considered.

To enable directional cloning, the forward PCR primer MUST contain thesequence, CACC, at the 5′ end of the primer. The four nucleotides, CACC,base pair with the overhang sequence, GTGG, in each pENTR TOPO® vector.

If the PCR product is to be expressed in mammalian cells (followingrecombination of the entry clone with a GATEWAY™ destination vector),the sequence of interest must include a Kozak translation initiationsequence with an ATG initiation codon for proper initiation oftranslation (Kozak, 1987; Kozak, 1991; Kozak, 1990). An example of aKozak consensus sequence is (G/A)NNATGG. Other sequences are possible,but the G or A at position −3 and the G at position +4 are the mostcritical for function (shown in bold). The ATG initiation codon is shownunderlined. Note: If the sequence of interest does not contain aninitiation codon within the context of a Kozak sequence, the forward PCRprimer may be designed so as to contain a Kozak sequence at the 5′ endof the primer (see below).

If the PCR product is to be expressed in prokaryotic cells without anN-terminal fusion tag (following recombination of the entry clone with aGATEWAY™-destination vector), the PCR product should be TOPO® Clonedinto a pENTR/SD/D-TOPO® entry vector. As noted above, pENTR/SD/D-TOPO®contains a T7 gene 10 translational enhancer and a ribosome binding site(RBS) to enable efficient translation of the PCR product in E. coli. Toensure optimal spacing for proper translation, the forward PCR primershould be designed such that that the ATG initiation codon of the PCRproduct directly follows the CACC necessary for directional cloning (seebelow).

Example of Forward Primer Design. Below is the DNA sequence of theN-terminus of a theoretical protein and the proposed sequence for acorresponding forward PCR primer. The ATG initiation codon isunderlined. DNA sequences:

(SEQ ID NO. 122) 5′-ATG GGA TCT GAT AAA Proposed Forward PCR primer:(SEQ ID NO. 123) 5′-CACC ATG GGA TCT GAT AAA.

If the forward PCR primer is designed as noted above, then (a) the ATGinitiation codon falls within the context of a Kozak sequence (see boxedsequence), allowing proper translation initiation of the PCR product inmammalian cells (note that the first three base pairs of the PCR productfollowing the 5′ CACC overhang will constitute a functional codon); and(b) the ATG initiation codon is properly spaced from the RBS (inpENTR/SD/D-TOPO® only), allowing proper translation of the PCR productin prokaryotic cells.

Guidelines to Design the Reverse primer. When designing your reverse PCRprimer, consider the following points below. See FIGS. 26 and 27 fordiagrams of the TOPO® Cloning sites for pENTR/D-TOPO® andpENTR/SD/D-TOPO®, respectively.

To ensure that the PCR product clones directionally with highefficiency, the reverse PCR primer MUST NOT be complementary to theoverhang sequence GTGG at the 5′ end. A one base pair mismatch canreduce the directional cloning efficiency from 90% to 50%, increasingthe likelihood that the ORF will be cloned in the opposite orientation(see “example A” below). We have not observed evidence of PCR productscloning in the opposite orientation from a two base pair mismatch.

If the PCR product is to be fused in frame with a C-terminal tag(following recombination of the entry clone with a GATEWAY™-destinationvector), then the reverse PCR primer should be designed so as to removethe native stop codon in the gene of interest (see “example B” below).

If the PCR product is NOT to be fused in frame with a C-terminal tag(following recombination of the entry clone with a GATEWAY™-destinationvector), then the native sequence containing the stop codon should beincluded in the reverse primer, or it should be ensured that the stopcodon is upstream from the reverse PCR primer binding site (see “exampleB” below).

Example A of Reverse Primer Design. Below is the sequence of theC-terminus of a theoretical protein. The protein should be fused inframe with a C-terminal tag (following recombination of the entry clonewith a GATEWAY™-destination vector). The stop codon is underlined.

DNA sequence: (SEQ ID NO. 46) AAG TCG GAG CAC TCG ACG ACG GTG TAG-3′.One solution is to design the reverse PCR primer to start with the codonjust upstream of the stop codon, but the last two codons contain GTGG(underlined below), which is identical to the 4 bp overhang sequence. Asa result, the reverse primer will be complementary to the 4 bp overhangsequence, increasing the probability that the PCR product will clone inthe opposite orientation. This situation should be avoided.

DNA sequence: (SEQ ID NO. 46) AAG TCG GAG CAC TCG ACG ACG GTG TAG-3′.Proposed Reverse PCR primer sequence: (SEQ ID NO. 47) TG AGC TGC TGC CAC AAA-5′.

Another solution is to design the reverse primer so that it hybridizesjust downstream of the stop codon, but still includes the C-terminus ofthe ORF. Note that the stop codon will need to be replaced with a codonfor an innocuous amino acid such as glycine, alanine, or lysine.

Example B of Reverse Primer Design. Below is the sequence for theC-terminus of a theoretical protein. The stop codon is underlined.

(SEQ ID NO. 48) GCG GTT AAG TCG GAG CAC TCG ACG ACT GCA TAG-3′.To fuse the ORF in frame with a C-terminal tag (supplied by thedestination vector after recombination), remove the stop codon bystarting with nucleotides homologous to the last codon (TGC) andcontinue upstream. The reverse primer will be:

(SEQ ID NO. 49) 5′-TGC AGT CGT CGA GTG CTC CGA CTT-3′.This will amplify the C-terminus without the stop codon and allow theORF to be joined in frame with a C-terminal tag. If it is not desirableto join the ORF in frame with a C-terminal tag, the reverse primershould simply be designed to include the stop codon:

(SEQ ID NO. 50) 5′-CTA TGC AGT CGT CGA GTG CTC CGA CTT-3′.Important: It must be remembered that the pENTR TOPO® vectors acceptblunt-end PCR products. 5′ phosphates should not be added to the primersfor PCR, as this will prevent ligation into the pENTR TOPO® vectors. Inaddition, it is recommended that the oligonucleotides be gel-purifiedprior to use, especially if they are long (>30 nucleotides).Producing Blunt-End PCR Products

Once a PCR strategy has been chosen and primers synthesized according tothe guidance presented above, the blunt-end PCR product can be produced.Any thermostable, proofreading polymerase may be used for this purpose,including ThermalAce™, PLATINUM®, Pfr, Pfu, or Vent® for PCR. To produceblunt-end PCR products, the instructions and recommendations of themanufacturer of the polymerase should be followed. It is important tooptimize PCR conditions to produce a single, discrete PCR product. Gelpurification of PCR fragments, according to methods outlined below, isalso recommended.

Producing PCR Products

To produce amplification products via PCR, 25 μl or 50 μl PCR reactionmixtures are set up using the following guidelines:

Follow the manufacturer's instructions for the DNA polymerase that isbeing used.

Use the cycling parameters suitable for the primers and template.

Use a 7 to 30 minute final extension to ensure that all PCR products arecompletely extended.

After cycling, the tube should be placed on ice or stored at −20° C. forup to 2 weeks.

Checking the PCR Product

To verify quality and quantity of the PCR product, 5 μl to 10 μl shouldbe removed from each PCR reaction and analyzed by agarose gelelectrophoresis for the following:

The presence of a single, discrete band of the correct size. If there isnot a single, discrete band, consult the manufacturer's recommendationsfor optimizing PCR reactions with the chosen polymerase. Alternatively,the desired product may be gel purified (see below).

Estimate the concentration of the PCR product. For TOPO® Cloning, a 5:1molar ratio of PCR product to TOPO® vector is recommended to obtain thehighest cloning efficiency. For example, 20 ng of a 500 bp PCR product,or 10 ng of a 1000 bp PCR product, may be used in a TOPO® Cloningreaction. The concentration of the PCR product may need to be adjustedbefore proceeding to TOPO® Cloning. Note: If ThermalAce™ polymerase isbeing used to produce the blunt-end PCR product, it should be noted thatThermalAce™ can generate higher yields than other proofreadingpolymerases. When generating PCR products in the 0.5 to 1.0 kb range, wegenerally dilute the PCR reaction 1:5 in 1× ThermalAce™ buffer beforeperforming the TOPO® Cloning reaction. For PCR products larger than 1.0kb, dilution may not be required.

Setting Up the TOPO® Cloning Reaction

Introduction

Once you have produced the desired PCR product, you are ready to TOPO®Clone it into the pENTR TOPO® vector and transform the recombinantvector into TOP10 E. coli. It is important to have everything you needset up and ready to use to ensure that you obtain the best possibleresults. We suggest that you read the sections entitled Setting Up theTOPO® Cloning Reaction and Transforming OneShot® TOP 10 Competent Cellsbefore beginning. If this is the first time you have TOPO® Cloned,perform the control reactions described below in parallel with yoursamples.

If you are TOPO® Cloning in HTP format (see below), you may transformTOP10 E. coli using Bulk TOP10 cells (500 reaction kits) or MultiShot™TOP10 cells (480 reaction kits). Depending on which kit you are using,see the TOPO® Cloning and transformation protocols below.

Note: Recent studies demonstrate that including salt (200 mM NaCl, 10 mMMgCl₂) in the TOPO® Cloning reaction may result in an increase in thenumber of transformants. From these results, we recommend adding salt tothe TOPO® Cloning reaction. A stock salt solution is provided in the kitfor this purpose. Please note that the amount of salt added to the TOPO®Cloning reaction varies depending on whether you plan to transformchemically competent cells or electrocompetent cells. For this reasontwo different TOPO® Cloning reactions are provided to help you obtainthe best possible results.

Transforming Chemically Competent E. coli

For TOPO® Cloning and transformation into chemically competent E. coli,adding sodium chloride and magnesium chloride to a final concentrationof 200 mM NaCl, 10 mM MgCl₂ in the TOPO® (Cloning reaction increases thenumber of colonies over time. A Salt Solution (1.2 M NaCl, 0.06 M MgCl₂)is provided to adjust the TOPO® Cloning reaction to the recommendedconcentration of NaCl and MgCl₂.

Transforming Electrocompetent E. coli

For TOPO® Cloning and transformation of electrocompetent E. coli, saltmay also be included in the TOPO® Cloning reaction, but the amount ofsalt must be reduced to 50 mM NaCl, 2.5 mM MgCl₂ to prevent arcing whenelectroporating. Dilute the Salt Solution 4-fold with water to prepare a300 mM NaCl, 15 mM MgCl₂ solution for convenient addition to the TOPO®Cloning reaction.

Setting Up the TOPO® Cloning Reaction

The table below describes how to set up your TOPO® Cloning reaction (6IA) for eventual transformation into either chemically competent OneShot! TOPO E. coli or electrocompetent E. coli. Additional informationon optimizing the TOPO® Cloning reaction for your needs can be foundbelow. If you generated your PCR product using ThermalAce-polymerase,please note that you may need to dilute your PCR reaction beforeproceeding.

Note: The blue color of the TOPO® vector solution is normal and is usedto visualize the solution.

TABLE 7 Setting Up a TOPO ® Cloning Reaction Mixture. ChemicallyCompetent Electrocompetent Reagents* E. coli E. coli Fresh PCR product0.5-4 μl 0.5-4 μl Salt Solution 1 μl — Dilute Salt Solution (1:4) — 1 μlSterile Water Add to final Add to final volume of 5 μl volume of 5 μlTOPO ® vector 1 μl 1 μl *Store all reagents at −20° C. when finished.Salt solutions and water can be stored at room temperature or 4° C.Performing the TOPO ® Cloning Reaction

Mix reaction gently and incubate for 5 minutes at room temperature(22-23° C.).

Note: For most applications, 5 minutes will yield plenty of colonies foranalysis. Depending on your needs, the length of the TOP011 Cloningreaction can be varied from 30 seconds to 30 minutes. For routinesubcloning of PCR products, 30 seconds may be sufficient. For large PCRproducts (>1 kb) or if you are TOPO® Cloning a pool of PCR products,increasing the reaction time may yield more colonies.

Place the reaction on ice and proceed to Transforming One Shot7 TOP10Competent Cells. Note: You may store the TOPO7 Cloning reaction at −20°C. overnight.

Transforming One Shot® TOP10 Competent Cells

Introduction

Once you have performed the TOPO® Cloning reaction, you will transformyour pENTR TOPO® construct into competent E. coli. One Shots TOP10Chemically Competent E. coli are included with the 20 reaction kit tofacilitate transformation, however, you may also transformelectrocompetent cells (see page x for ordering information). Protocolsto transform chemically competent or electrocompetent E. coli areprovided in this section.

Materials Supplied by the User

In addition to general microbiological supplies (i.e. plates,spreaders), you will need the following reagents and equipment.

-   -   (a) 42° C. water bath (or electroporator with cuvettes,        optional)    -   (b) LB plates containing 50 μg/ml kanamycin (two for each        transformation)    -   (c) 37° C. shaking and non-shaking incubator        There is no blue-white screening for the presence of inserts.        Most transformants will contain recombinant plasmids with the        PCR product of interest cloned in the correct orientation.        Sequencing primers are included in the kit to sequence across an        insert in the multiple cloning site to confirm orientation and        reading frame.        Preparing for Transformation

For each transformation, you will need one vial of competent cells andtwo selective plates.

Equilibrate a water bath to 42° C. (for chemical transformation) or setup your electroporator if you are using electrocompetent E. coli.

For electroporation, dilute a small portion of the Salt Solution 4-foldto prepare Dilute Salt Solution (e.g. add 5 μl of the Salt Solution to150 sterile water).

Warm SOC medium to room temperature.

Warm LB plates containing 50 μg/ml kanamycin at 37° C. for 30 minutes.

Thaw on ice 1 vial of One Shot® TOP 10 cells for each transformation.

Important: Please note that directional TOPO® Cloning generally yields 5to 10-fold fewer colonies than traditional bidirectional TOPO TACloning®. When directionally TOPO® Cloning a 750 bp test insert, wegenerally obtain 1800-3000 colonies using the protocol described herein.Although fewer total colonies are obtained, greater than 90% of thecolonies will contain plasmid with your PCR insert in the correctorientation.

One Shot® TOP10 Chemical Transformation Protocol

1. Add 2 μl of the TOPO® Cloning reaction from Performing the TOPO®Cloning Reaction (above) into a vial of One Shot TOP10 ChemicallyCompetent E. coli and mix gently. Do not mix by pipetting up and down.

2. Incubate on ice for 5 to 30 minutes.

Note: Longer incubations on ice seem to have a minimal effect ontransformation efficiency. The length of the incubation is at the user'sdiscretion.

3. Heat-shock the cells for 30 seconds at 42° C. without shaking

4. Immediately transfer the tubes to ice.

5. Add 250 μl of room temperature SOC medium.

6. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C. for 30 minutes.

7. Spread 50-200 μl from each transformation on a prewarmed selectiveplate and incubate overnight at 37° C. We recommend that you plate twodifferent volumes to ensure that at least one plate will havewell-spaced colonies.

8. An efficient TOPO7 Cloning reaction may produce several hundredcolonies. Pick .about.5 colonies for analysis (see AnalyzingTransformants, below).

Transformation by Electroporation

Use ONLY electrocompetent cells for electroporation to avoid arcing. Donot use the One Shot® TOP10 chemically competent cells forelectroporation.

1. Add 2 μl of the TOPO® Cloning reaction from Performing the TOPO®Cloning Reaction (above) into a 0.1 cm cuvette containing 50 μl ofelectrocompetent E coli and mix gently. Do not mix by pipetting up anddown. Avoid formation of bubbles.

2. Electroporate your samples using your own protocol and yourelectroporator.

Note: If you have problems with arcing, see below.

3. Immediately add 250 μl of room temperature SOC medium.

4. Transfer the solution to a 15 ml snap-cap tube (i.e. Falcon) andshake for at least 1 hour at 37° C. to allow expression of the kanamycinresistance gene.

5. Spread 20-100 μl from each transformation on a prewarmed selectiveplate and incubate overnight at 37° C. To ensure even spreading of smallvolumes, add 20 μl of SOC. We recommend that you plate two differentvolumes to ensure that at least one plate will have well-spacedcolonies.

6. An efficient TOPO7 Cloning reaction may produce several hundredcolonies. Pick .about.5 colonies for analysis (see AnalyzingTransformants, below).

Addition of the Dilute Salt Solution in the TOPO® Cloning Reactionbrings the final concentration of NaCl and MgCl₂ in the TOPO® CloningReaction to 50 mM and 2.5 mM, respectively. To prevent arcing of yoursamples during electroporation, the volume of cells should be between 50and 80 μl (0.1 cm cuvettes) or 100 to 200 μl (0.2 cm curettes).

If you experience arcing during transformation, try one of the followingsuggestions:

Reduce the voltage normally used to charge your electroporator by 10%

Reduce the pulse length by reducing the load resistance to 100 ohms

Ethanol precipitate the TOPO® Cloning reaction and resuspend in waterprior to electroporation.

High-Throughput Applications

The 480 and 500 reaction pENTR and pENTR/SD Directional TOPO® CloningKits are specifically designed to allow production of GATEWAY™ entryclones for use in high-throughput (HTP) applications. In these kits, thepENTR TOPO® vector is provided in bulk and chemically competent TOP10 E.coli are provided in a choice of two formats:

Cells are provided in bulk aliquots of 5 ml to allow simple transfer ofthe cells from a sterile trough into a 96-well plate containing theTOPO7 Cloning reaction (Catalog nos. K2400-500 and K2420-500).

Cells are provided pre-aliquoted in 96-well plates (in 12-wellstripwells) to allow addition of the TOPO7 Cloning reaction to the cells(Invitrogen Corporation, Carlsbad, Calif.; Catalog nos. K2400-480 andK2420-480).

HTP TOPO® Cloning and Transformation with Bulk Cells

Description

In this protocol, the TOPO® Cloning reaction is set up in a 96-well Ubottom, polystyrene plate (Costar, Catalog no. 3366, 330 μl/well) andthe TOP10 competent cells are placed in a trough for dispensing.

Before Starting

Chill a 96-well metal heating block (VWR, Catalog no. 13259-260) on iceuntil the block is cold.

Bring a vial of SOC to room temperature.

Pre-heat a heat block or thermocycler containing a 96-well metal blockto 42° C.

Note: You can also use a water bath, but be careful not to contaminatethe cells.

-   -   Thaw 1 tube (5 ml) of TOP10 chemically competent E. coli on ice        (30-60 minutes).    -   Warm LB agar plates containing 50 μg/ml kanamycin to 37° C. If        you plan to include a pUC19 control to test the transformation        efficiency of the cells, you will need LB agar plates containing        50-100 μg/ml ampicillin. Controls: For your convenience a 50 μl        aliquot of competent cells is provided to perform a test TOPO®        Cloning and transformation reaction. In addition, you can        include the pUC19 plasmid as an internal control (see Procedure        below).

Procedure

1. Set up the 6 μl TOPO® Cloning reaction in each well as follows. Ifyou include pUC19 as a control, leave 2-3 wells empty.

PCR product 1 μl

Salt Solution 1 μl

Sterile Water 3 μl

pENTR TOPO® vector 1 μl

Final Volume 6 μl

2. Incubate 5-10 minutes at room temperature.

3. Place the 96-well plate on the cooling block for 5 minutes.

4. If you are including pUC19, add 1 μl (10 pg) of the plasmid to 2-3empty wells.

5. Pour thawed TOP10 E. coli into a sterile trough and immediatelydispense 45 μl/well. Gently pipette up and down 1-2 times to mix.

6. Cover the plate with Parafilm® and incubate it on the chilled blockfor 20 minutes.

7. Transfer the plate to either the pre-warmed heat block or thethermocycler and heat-shock the cells at 42° C. for 30 seconds.

8. Transfer the plate back to the cooling block and press down to ensurethe plate is in complete contact with the cooling block. Incubate for 1minute.

9. Remove the Parafilm® and add 150 μL/well of SOC.

10. Re-cover the plate and incubate the plate at 37° C. for 1 hour.Note: Gentle shaking (125 RPM) is optional.

11. Plate 50 μL from each well onto LB agar plates containing 50 μg/mlkanamycil. For the pUC19 controls, plate 10 μl of the transformationmixture plus 20 μl of SOC on LB plates containing 100 μg/ml ampicillin.Incubate overnight at 37° C.

12. The next day, select 5-10 colonies and process as desired.

Too Many Colonies

If you obtain too many colonies, reduce the amount of bacterial cultureplated and/or dilute the transformation with additional SOC.

HTP TOPO Cloning and Transformation with MultiShot™ Cells

Description

In this protocol, the TOPO® Cloning reaction is set up in a 96-wellplate and 2 μl are transferred to each well of a 96-well MultiShot™plate containing 15 μl of chemically competent TOP 10 E. coli per well.

Before Starting

-   -   Chill two 96-well metal heating blocks (VWR, Catalog no.        13259-260) on ice until the blocks are cold.    -   Bring a vial of SOC to room temperature.    -   Warm LB agar plates containing 50 μg/ml kanamycin to 37° C. If        you plan to include a pUC19 control to test the transformation        efficiency of the cells, you will need LB agar plates containing        50-100 μg/ml ampicillin.    -   Pre-heat a heat block or thermocycler containing a 96-well metal        block to 42° C.    -   Note: You can also use a water bath, but be careful not to        contaminate the cells.    -   If you are using a thermocycler, program the machine to hold the        temperature at 42° C.

Controls: A test plate containing 1 row (12 wells) of TOP10 cells isincluded to perform test TOPO® Cloning reactions and transformations. Inaddition, you can include the pUC19 plasmid as an internal control (seeProcedure below).

Procedure

1. In a 96-well plate, set up the following 6 μl TOPO® Cloning reactionin each well.

PCR product 1 μl

Salt Solution 1 μl

Sterile Water 3 μl

pENTR TOPO® vector 1 μl

Final Volume 6 μl

2. Incubate 5-10 minutes at room temperature.

3. Place the 96-well plate on one of the cooling blocks for 5 minutes.

4. Remove a 96-well MultiShot™ plate of chemically competent TOP10 E.coli from the freezer and place it in the second cooling block. Cellsshould thaw within 30 seconds.

5. Carefully remove the aluminum foil seal.

6. Use a multi-channel pipette to add 2 μl of each TOPO® Cloningreaction (.about.3.3 ng) to each well of the 96-well plate containingcells. Keep the volume around 2 μl for uniform results. For the pUC19control, add 1 μl (10 pg) of the DNA.

7. Cover the cells with the supplied plastic lid and incubate the cellsand DNA in the chilled block for 20 minutes.

8. Transfer the cell plate to either the pre-warmed heat block orthermocycler and heat-shock for 30 seconds at 42° C.

9. Transfer the cell plate back to a cooling block, press the plate intothe block and allow the plate to cool for 1 minute.

10. Remove the plastic lid and add 90 μl SOC to each well.

11. Cover the plate with the lid and incubate the plate at 37° C. for 1hour. Note: Gentle shaking (125 RPM) is optional.

12. Plate 100 μl from each well onto LB agar plates containing 50 μg/mlkanamycin. For the pUC19 controls, plate 10 μl of the transformationmixture plus 20 μl of SOC on LB plates containing 100 μg/ml ampicillin.Incubate overnight at 37° C.

NOTE: If you obtain too many colonies, you can reduce the amount ofcells plated or dilute the TOPO® Cloning reactions with sterile water orTE buffer prior to adding the reaction to the cells.

Analyzing Transformants

Analyzing Positive Clones

1. Pick 5 colonies and culture them overnight in LB or SOB mediumcontaining 50-100 μg/ml kanamycin.

2. Isolate plasmid DNA using your method of choice. If you needultra-pure plasmid DNA for automated or manual sequencing, we recommendusing the S.N.A.P.J MidiPrep Kit (Catalog no. K1910-01).

3. Analyze the plasmids by restriction analysis to confirm the presenceand correct orientation of the insert. Use a restriction enzyme or acombination of enzymes that cut once in the vector and once in theinsert.

Sequencing

You may sequence your construct to confirm that your gene is cloned inthe correct orientation. The M13 Forward (−20) and M13 Reverse primersare included in the kit to help you sequence your insert. The M13Forward (−20) and M13 Reverse primers are also available separately fromInvitrogen Corporation, Carlsbad, Calif.

Important: If you download the sequence for pENTR/D-TOPO® orpENTR/SD/D-TOPO® from the Invitrogen Corporation Web site (seedescription for FIG. 22), note that the overhang sequence (GTGG) will beshown already hybridized to CACC. No DNA sequence analysis programallows us to show the overhang without the complementary sequence.

Analyzing Transformants by PCR

You may analyze positive transformants using PCR. For PCR primers, use acombination of the M13 Forward (−20) primer or the M13 Reverse primerand a primer that hybridizes within your insert. You will have todetermine the amplification conditions. If you are using this techniquefor the first time, we recommend performing restriction analysis inparallel. Artifacts may be obtained because of mispriming orcontaminating template.

The protocol below is provided for your convenience. Other protocols aresuitable.

1. Prepare a PCR cocktail consisting of PCR buffer, dNTPs, primers, andTaq polymerase. Use a 20 μl reaction volume. Multiply by the number ofcolonies to be analyzed (e.g. 5).

2. Pick 5 colonies and resuspend them individually in 20 μl of the PCRcocktail (remember to make a patch plate to preserve the colonies forfurther analysis).

3. Incubate reaction for 10 minutes at 94° C. to lyse cells andinactivate nucleases.

4. Amplify for 20 to 30 cycles.

5. For the final extension, incubate at 72° C. for 10 minutes. Store at−4° C.

6. Visualize by agarose gel electrophoresis.

Important: If you have problems obtaining transformants or the correctinsert, perform the control reactions described herein. These reactionswill help you troubleshoot your experiment.

Long-Term Storage

Once you have identified the correct clone, be sure to purify the colonyand make a glycerol stock for long term storage. We recommend that youstore a stock of plasmid DNA at −20° C.

1. Streak the original colony out for single colony on LB platescontaining 50 μg/ml kanamycin.

2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50μg/ml kanamycin.

3. Grow until culture reaches stationary phase.

4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transferto a cryovial.

5. Store at −80° C.

Recombining the Entry Construct with a Destination Vector

Once you have obtained your entry clone, you may recombine the pENTRTOPO® construct with any GATEWAY™ destination vector of choice togenerate an expression clone. This “LR” recombination reaction ismediated by LR CLONASE™, a cocktail of recombination proteins. LRCLONASE™ Enzyme Mix is available from Invitrogen Corporation (Carlsbad,Calif.). In certain such methods, for example, TOPO-adapted vectors areincubated with one or more nucleic acid segments (e.g., one or more PCRproducts) at room temperature (e.g., about 20-20° C.) for about 5-30(and preferably about 10) minutes; the reaction is then heat-treated byincubation at about 80° C. for about 20 minutes, and the reactionmixture then used in a standard LR reaction according to manufacturer'sinstructions (Invitrogen Corporation, Carlsbad, Calif.), except theincubation time for the LR reaction is increased to about 3 hours.

Optimizing the TOPO® Cloning Reaction

Speeding up the Cloning Process. The high efficiency of TOPO® Cloningallows you to streamline the cloning process. If you routinely clone PCRproducts and wish to speed up the process, consider the following:

-   -   Incubate the TOPO® Cloning reaction for only 30 seconds instead        of 5 minutes.

You may not obtain the highest number of colonies, but with the highefficiency of TOPO® Cloning, most of the transformants will contain yourinsert.

-   -   After adding 3 μl of the TOPO® Cloning reaction to chemically        competent cells, incubate on ice for only 5 minutes.

Increasing the incubation time to 30 minutes does not significantlyimprove transformation efficiency.

Obtaining More Transformants. If you are TOPO® Cloning large PCRproducts, toxic genes, or cloning a pool of PCR products, you may needmore transformants to obtain the clones you want. To increase the numberof colonies:

-   -   Incubate the salt-supplemented TOPO® Cloning reaction for 20 to        30 minutes instead of 5 minutes.

Increasing the incubation time of the salt-supplemented TOPO® Cloningreaction allows more molecules to ligate, increasing the transformationefficiency. Addition of salt appears to prevent topoisomerase I fromrebinding and nicking the DNA after it has ligated the PCR product anddissociated from the DNA.

-   -   Titrate the amount of PCR product used in the TOPO7 Cloning        reaction for maximum colony output.

Cloning Dilute PCR Products

To clone dilute PCR products, you may:

-   -   Increase the amount of the PCR product    -   Incubate the TOPO® Cloning reaction for 20 to 30 minutes    -   Concentrate the PCR product

Performing the Control Reactions

Introduction

We recommend performing the following control TOPO® Cloning reactionsthe first time you use the 20 reaction kit to help you evaluate yourresults. Performing the control reactions involves producing a controlPCR product using the reagents included in the kit and using thisproduct directly in a TOPO® Cloning reaction.

Before Starting

For each transformation, prepare two LB plates containing 50 μg/mlkanamycin.

Producing the Control PCR Product

Use your thermostable, proofreading polymerase and the appropriatebuffer to amplify the control PCR product. Follow the manufacturer'srecommendations for the polymerase you are using.

1. To produce the 750 bp control PCR product, set up the following 50 μlPCR:

Control DNA Template (100 ng) 1 μl

10×PCR Buffer (appropriate for enzyme) 5 μl

dNTP Mix 0.5 μl

Control PCR Primers (0.1 μg/μl each) 1 μl

Sterile Water 41.5 μl

Thermostable polymerase (1-2.5 units/μl) 1 μl

Total Volume 50 μl

2. Overlay with 70 μl (1 drop) of mineral oil.

3. Amplify using the following cycling parameters:

Stop Time Temperture Cycles Initial Denaturation 2 minutes 94° 1XDenaturation 1 minutes 94° Annealing 1 minutes 55° Extension 1 minutes72° 25X  Final Extension 7 minutes 72° 1X

4. Remove 10 μl from the reaction and analyze by agarose gelelectrophoresis. A discrete 750 bp band should be visible. Proceed tothe Control TOPO7 Cloning Reactions.

Control TOPO® Cloning Reactions

Using the control PCR product produced on the previous page and thepENTR is TOPO® vector, set up two 6 μl TOPO® Cloning reactions asdescribed below.

1. Set up control TOPO® Cloning reactions: 14 Reagent “Vector Only”“Vector+PCR Insert” Sterile Water 4 μl 3 μl Salt Solution or Dilute 1 μl1 μl Salt Solution Control PCR Product—1 μl pENTR TOPO® 1 μl 1 μl vector

Reagent “Vector Only” “Vector + PCR Insert” Sterile Water 4 μl 3 μl SaltSolution or Dilute Salt 1 μl 1 μl Solution Control PCR Product — 1 μlpENTER TOPO vector 1 μl 1 μl

2. Incubate at room temperature for 5 minutes and place on ice.

3. Transform 3 μl of each reaction into separate vials of One Shot®TOP10 cells.

4. Spread 100-200 μl of each transformation mix onto LB platescontaining 50 μg/ml kanamycin. Be sure to plate two different volumes toensure that at least one plate has well-spaced colonies.

5. Incubate overnight at 37° C.

Analysis of Results

Hundreds of colonies from the vector+PCR insert reaction should beproduced. To analyze the transformations, isolate plasmid DNA and digestwith the appropriate restriction enzymes. Greater than 90% of thecolonies should contain the 750 bp insert in the correct orientation.Relatively few colonies should be produced in the vector-only reaction.

Transformation Control

pUC19 plasmid is included to check the transformation efficiency of theOne Shot® TOP10 competent cells. Transform one vial of One Shot® TOP10cells with 10 pg of pUC19 using the protocol described above. Plate 10μl of the transformation mixture plus 20 μl of SOC on LB platescontaining 100 μg/ml ampicillin. Transformation efficiency should be−1×10⁹ cfu/μg DNA.

Factors Affecting Cloning Efficiency

Please note that lower cloning efficiencies will result from thefollowing variables. Most of these are easily corrected, but if you arecloning large inserts, you may not obtain the expected 90% directionalcloning efficiency.

Variable Solution Low efficiency of Forward primner should contain CACCat directional cloning 5′ end. Reverse primer is complementary to theoverhang at the 5′ end. Re-design primer to avoid base pairing to theoverhang. pH > 9 in PCR Check the pH of the PCR amplificationamplification reaction reaction and adjust with 1M Tris-HCl, pH 8.Incomplete extension Be sure to include a final extension step of duringPCR 7 to 30 minutes during PCR. Longest PCR products will need a longerextension time. Cloning large inserts Increase amount of insert orgel-purify as (>1 kh) described on pages 25-26. Excess (or overlydilute) Reduce (or concentrate) the amount of PCR PCR product product.PCR cloning artifacts TOPO ® Cloning is very efficient for small (“falsepositives”) fragments (<100 bp) present in certain PCR reactions.Gel-purify your PCR product or optimize your PCR.

Gel Purifying PCR Products

Introduction

Smearing, multiple banding, primer-dimer artifacts, or large PCRproducts (>3 kb) may necessitate gel purification. If you wish to purifyyour PCR product, be extremely careful to remove all sources of nucleasecontamination. There are many protocols to isolate DNA fragments orremove oligonucleotides. Please refer to Current Protocols in MolecularBiology, Unit 2.6 (Ausubel et al., 1994) for the most common protocols.Three simple protocols are provided below.

Note: cloning efficiency may decrease with purification of the PCRproduct (e.g. PCR product too dilute). You may wish to optimize your PCRto produce a single band (see Producing Blunt-End PCR Products, herein).

Using the S.N.A.P.™ Gel Purification Kit

The S.N.A.P.™ Gel Purification Kit available from InvitrogenCorporation, Carlsbad, Calif. (Catalog no. K1999-25) allows you torapidly purify PCR products from regular agarose gels.

1. Electrophorese amplification reaction on a 1 to 5% regular TAEagarose gel. (Note: Do not use TBE to prepare agarose gels. Borateinterferes with the sodium iodide step, below.)

2. Cut out the gel slice containing the PCR product and melt it at 65°C. in 2 volumes of the 6 M sodium iodide solution.

3. Add 1.5 volumes Binding Buffer.

4. Load solution (no more than 1 ml at a time) from Step 3 onto aS.N.A.P.™ column. Centrifuge 1 minute at 3000×g in a microcentrifuge anddiscard the supernatant.

5. If you have solution remaining from Step 3, repeat Step 4.

6. Add 900 μl of the Final Wash Buffer.

7. Centrifuge 1 minute at full speed in a microcentrifuge and discardtile flowthrough.

8. Repeat Step 7.

9. Elute the purified PCR product in 40 μl of TE or sterile water. Use 4μl for the TOPO® Cloning reaction and proceed as described above.

Quick S.N.A.P.™ Method

An even easier method is to simply cut out the gel slice containing yourPCR product, place it on top of the S.N.A.P.™ column bed, and centrifugeat full speed for 10 seconds. Use 1-2 μl of the flow-through in theTOPO® Cloning reaction. Be sure to make the gel slice as small aspossible for best results.

Low-Melt Agarose Method

If you prefer to use low-melt agarose, use the procedure below. Pleasenote that gel purification will result in a dilution of your PCR productand a potential loss of cloning efficiency.

1. Electrophorese as much as possible of your PCR reaction on a low-meltagarose gel (0.8 to 1.2%) in TAE buffer.

2. Visualize the band of interest and excise the band.

3. Place the gel slice in a microcentrifuge tube and incubate the tubeat 65° C. until the gel slice melts.

4. Place the tube at 37° C. to keep the agarose melted.

5. Add 4 μl of the melted agarose containing your PCR product to theTOPO® Cloning reaction as described above.

6. Incubate the TOPO® Cloning reaction at 37° C. for 5 to 10 minutes.This is to keep the agarose melted.

7. Transform 2 to 4 μl directly into OneShot® TOP10 cells using themethod on page 13.

Note: the cloning efficiency may decrease with purification of the PCRproduct. You may wish to optimize your PCR to produce a single band.

Example 9 Optimization of Reaction Conditions for TOPO Joining ReactionsUsing GATEWAY™ Vectors

To use TOPO Cloning procedures in conjunction with GATEWAY vectors, theoptimal conditions for the combined reactions were investigated. Incarrying out these studies, several questions were addressed.

Sufficiency of Template for BP Reaction, and Inhibition of BP Reactionby TOPO Reaction Components

To address these issues, TOPO Tools was used as described elsewhereherein to generate attB1+CAT+attB2 templates. Secondary PCR was thenperformed to generate sufficient template for testing studies, and BPreactions were performed using the products. The following reactionconditions were used for each step of the process:

TOPO Joining Reaction: BP Reaction: X ng of PCR product (see below) 2 μlsalt-free buffer 1 μl topoisomerase 1 μl TOPO Joining Product 0.5 μl of500 mM Tris 0.5 μl of pDONR222 (300 ng/μl) 1 μl of 40 mM NaCl 2 μl of BPClonase (Invitrogen Corporation, Carlsbad, CA) 37° C. for 15 min roomtemp for 25 min → Proteinase Transformation (chemical) K treatment

Following BP reactions, mixtures were chemically transformed intochemically competent E. coli cells (e.g., TOP10; Invitrogen CorporationCarlsbad, Calif.) and cells were plated to determine recombinationefficiency.

Results

1 2 3 4 5 6 Colonies 149 270 514 0 0 0 Template 0.8 ng 1.6 ng 4 ng 1.6ng 4 ng 0 ng Used TOPO No No No Yes Yes No Joining?

These results demonstrate that TOPO Tools generates sufficient templatefor the subsequent BP reaction. In addition, these results demonstratethat TOPO joining inhibits the subsequent BP reaction.

Effect of Presence of attB1 and attB2 Adapters on BP Reactions

In this portion of the studies, the effects of the presence of excessattB1 and attB2 adapters in the reaction mixtures on the subsequent BPreaction were examined. To address this issue, different amounts ofattB1 and attB2 adapters were added to templates (attB1+CAT+attB2, 20ng), and BP reactions were performed under standard conditions (60minutes at room temperature). Following BP reactions, mixtures werechemically transformed into chemically competent E. coli cells (e.g.,TOP10; Invitrogen Corporation, Carlsbad, Calif.) and cells were platedto determine recombination efficiency.

Results:

1 2 3 4 5 6 Adaptor amount (ng) 20 10 5 2.5 1 0 No. of colonies formed270 475 760 590 340 460

These results demonstrate that the presence of an excess of attB1 andattB2 adapters has no significant effect on the transformationefficiencies observed, indicating that the BP reaction is notsignificantly influenced by the presence of attB1 and attB2 adapters inthe reaction mixture.

Removal of Inhibitors from TOPO Joining Reactions

To address the optimal methods for removing inhibitors from TOPO Joiningreactions prior to use of the products in BP reactions, varioustreatment methods were assessed. TOPO Joining reactions were performedusing the following reaction mixtures, incubated at room temperature for5 minutes:

attB1 + attB2 (20 ng/μl each) 2 μl CAT (100 ng/μl) 1.7 μl attB1 + CAT +attB2 product (10 ng/μl) 1 μl 500 mM Tris 0.5 μl Topoisomerase (1 μg/μl)1 μl

Following TOPO Joining reactions, seven different samples of thereaction mixtures were treated under one of the following conditionsprior to carrying out BP reactions:

(1) add 1 μl of 0.6% SDS+3 mM EDTA to one reaction, 37° C. for 15 min;

(2) add 4 μl of 0.6% SDS+3 mM EDTA to four reactions, 37° C. for 15 min,then SNAP purify into 20 μl of water;

(3) add 4 μl of 0.6% SDS+3 mM EDTA+1 μl of proteinase K (2 μg/μl) to 4reactions, 37° C. for 15 minutes, then SNAP purify into 20 μl of water;

(4) add 0.8 μl of 2.5 M NaCl to one reaction, 37° C. for 17 minutes;

(5) add 3.2 μl of 2.5 M NaCl to four reactions, 37° C. for 15 min, thenSNAP purify into 20 μl of water;

(6) add 3.2 μl of 2.5 M NaCl and 1 μl of 2 μg/μl proteinase K to 4reactions, 37° C. for 15 min, then SNAP purify into 20 μl of water(positive control; 0.8 ng template used);

(7) (negative control; no template used).

BP reactions were performed using salt-free buffer for 60 min at roomtemperature. For unpurified mixtures, 1 μl of TOPO Joining reactionmixture was used per 10 μl of BP reaction. For purified mixtures, 5.5 μlof TOPO Joining reaction mixture was used per 10 μl of BP reaction.Following BP reactions, mixtures were chemically transformed intochemically competent E. coli cells (e.g., TOP10; Invitrogen Corporation,Carlsbad, Calif.) and cells were plated to determine recombinationefficiency.

Results

1 2 3 4 5 6 7 8 Treatment SDS SDS SDS NaCl NaCl NaCl (+) (−) ProteinaseK − − + − − + Purification − + + − + + No. of Colonies 6 515 400 0 550657 179 0

These results demonstrate that: (1) purification is not necessary tocarry out the BP reaction efficiently; (2) treatment of reactionmixtures with proteinase K is not required following TOPO Joiningreactions for maximum efficiency of subsequent BP reactions; and (3) SDStreatment and NaCl treatment of reaction mixtures give the sametransformation efficiencies (and therefore have the same effects uponthe BP reaction).

Optimization of BP Reaction Temperature

To determine the optimum reaction temperature for carrying out BPreactions following TOPO Joining, attB1+CAT+attB2 PCR product was usedas the template for BP reactions conducted under various temperatures.Following BP reactions, mixtures were chemically transformed intochemically competent E. coli cells (e.g., TOP10; Invitrogen Corporation,Carlsbad, Calif.) and cells were plated to determine recombinationefficiency.

Results

BP Reaction Temperature Room 42° C. 37° C. Temp 14° C. No. of Colonies(+Template) 3 337 588 195 No. of Colonies (no Template) 0 4 0 0

These results demonstrate that room temperature (about 20-25° C.) is theoptimal reaction temperature for carrying out BP reactions.

Optimization of Molar Ratio of attB1:insert:attB2

To determine the optimal molar ratio for attB1, insert and attB2templates in the BP reaction, these templates were mixed in variousmolar ratios and BP reactions carried out under optimal conditionsdescribed above. Following BP reactions, mixtures were chemicallytransformed into chemically competent E. coli cells (e.g., TOP10;invitrogen Corporation, Carlsbad, Calif.) and plated to determinerecombination efficiency.

Results

Ratio of attB1:insert:attB2 2:1:2 1.5:1:1.5 1:1:1 1:2:1 0 (control) No.of Colonies 81 93 165 154 9

These results demonstrate that a ratio of attB1:insert:attB2 at 1:1:1 isoptimal for carrying out BP reactions.

Determination of Effect of Salt on BP Reaction

To determine whether the presence of salt in the BP reaction solutioninfluences the recombination efficiency, BP reactions were carried outin salt-free buffers, or in standard BP reaction buffers containingsalt.

Results

Buffer Salt − + +template 108 109 −template (neg. control) 1 0

These results demonstrate that the presence or absence of salt in thereaction buffer during the BP reaction has no impact upon therecombination efficiency.

Determination of Optimal Number of TOPO Joining Reactions

In the next series of studies, the question of whether one TOPO Joiningreaction is sufficient to provide optimal recombination efficiency forBP reactions after purification was examined. A single TOPO Joiningreaction was carried out using the following reaction mixture:

24 attB1 and attB2 (20 ng/μl each) 0.5 μl CAT (100 ng/μl) 1.7 μl 500 mMTris 0.5 μl Topoisomerase (1 μg/μl) 1 μl dH₂O sufficient to bring finalvolume to 5 μl

The reaction mixture was incubated at 37° C. for 15 minutes, then 1 μlof 0.6% SDS+3 mM EDTA was added; the mixture was incubated at 37° C. for15 minutes, and then purified using a SNAP column (see above) into 20 μlof water. A BP reaction was then carried out using the product of thisTOPO Joining reaction as follows:

standard BP reaction buffer 2 μl pDONR222 (300 ng/μl) 0.5 μl TOPOJoining product (from above) 5.5 μl BP Clonase 2 μl

The reaction mixture was incubated at room temperature for 60 minutes,then 1 μl of 2 μg/μl proteinase K was added; the mixture was incubatedat 37° C. for 15 minutes, and then at 75° C. for 15 minutes. 4 μl ofthis reaction mixture was then used for chemical transformation intochemically competent E. coli cells (e.g., TOP10; Invitrogen Corporation,Carlsbad, Calif.) and cells were then plated to determine recombinationefficiency.

Results (No. of Colonies Formed):

+Templates −Template (neg. control) 188 0

These results demonstrate that one TOPO Joining reaction providessufficient template to carry out an efficient BP reaction.

Optimization of Purification Methods

Studies were also conducted to determine whether the SNAP purificationcolumn (Invitrogen Corporation, Carlsbad, Calif.) or the CONCERTpurification system (Invitrogen Corporation, Carlsbad, Calif.) differedin providing optimal purified template for carrying out BP reactionsafter TOPO Joining TOPO Joining reactions and BP reactions wereconducted as described above, except that some samples were purifiedusing SNAP columns, and other samples were purified using the CONCERTplasmid purification system after conducting the TOPO Joining reaction.Purified samples were then carried through a standard BP reaction, andreaction mixtures were then used either for transformation via chemicaltransformation or electroporation. Following transformation, cells wereplated to determine recombination efficiency.

Results (No. of Colonies Formed)

Transformation Method SNAP Concert No template (neg. control) Chemical188 254 0 Electroporation 8220 11,460 672

These results demonstrate that both SNAP and CONCERT purificationsystems work well to provide purified template for BP reactions afterTOPO Joining reactions.

Optimal Conditions

Based on the results of the above studies taken together, it wasdetermined that the optimal conditions for combination TOPOJoining-Gateway reactions are as follows:

(1) TOPO Joining Reaction

(a) attB1/insert at 1:1 molar ratio, in 5 μl reaction volume

(b) incubate at 37° C. for 15 minutes

(c) add 1 μl of 0.6% SDS+3 mM EDTA; incubate at 37° C. for 15 minutes

(d) purify with SNAP column or CONCERT system into 20 μl of dH₂O

(2) BP Reaction

(a) prepare reaction mixture: 28 (i) purified TOPO Joining product 5.5μl; (ii) standard BP reaction buffer 2 μl; (iii) pDONR222 (30 ng/μl) 0.5μl; (iv) BP Clonase 2 μl;

(b) incubate reaction mixture at room temperature for 60 minutes;

(c) add 1 μl of 2 μg/μl proteinase K;

(d) incubate at 37° C. for 15 minutes;

(e) incubate at 75° C. for 15 minutes;

(3) Transformation

(a) use 2-4 μl of reaction mixture from BP reaction, and carry outeither chemical transformation or electroporation.

To demonstrate the efficacy of these optimized conditions, studies wereconducted using CAT and lacZ inserts of various sizes subjected to TOPOJoining and subsequent BP reactions, followed by transformation andplating.

Results

Chemical Transformation

lacZ lacZ lacZ lacZ Insert CAT (1 kb) (1.5 kb) (2 kb) (3.2 kb) none No.of Colonies 188 180 182 177 71 3 Right-sized Clone 10/10 18/18 16/1617/18 18/18 —

Electrical Transformation

lacZ lacZ lacZ lacZ Insert CAT (1 kb) (1.5 kb) (2 kb) (3.2 kb) none No.of Colonies 8222 7335 7320 7500 6150 510

These results, taken together, demonstrate that the conditions describedabove are optimal for combination TOPO Joining-Gateway reactions oninserts of various sizes.

Example 10 Construction of a Mammalian Expression Cassette withoutSecondary PCR Methods

Preparation of Elements and Gene of Interest

The following primer sets (see Table 8 below) and templates were usedfor PCR amplification of elements and gene of interest:

(A) Primer set: Sequence #1 and #2; template: pcDNA 4/TetO. PCR product:5′ element.

(B) Primer set: Sequence #3 and #4; template: pcDNA 3.2N5. PCR product:3′ element.

(C) Primer set: Sequence #5 and #6; template: pcDNA 3.1/CAT. PCRproduct: CAT insert.

TABLE 8 Primers Used for Construction of Expression Cassette. SEQ ID NO:51 GTTGACATTGATTATTGACTAG SEQ ID NO: 52 GTTCCGAAGGGTTAACGCTAGAGTCCGGAGGCSEQ ID NO: 53 GACTCAAAGGGAAGGTAAGCCTATCCCTAAGG SEQ ID NO: 54GCGCAGATCTGCTATGGCAG SEQ ID NO: 55 CGGAACAAGGGACCATGGAGAAAAAAATCACTGGATASEQ ID NO: 56 TGAGTCAAGGGCGCCCCGCCCTGCTGCCACTCATCG SEQ ID NO: 57GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCCTTCG GAAC SEQ ID NO: 58GTTCCGAAGGGAAGCCTGCTTTTTTGTACAAACTTGT CCCC SEQ ID NO: 59GAGTCAAAGGGACCCAGCTTTCTTGTACAAAGTGGTC CCC SEQ ID NO: 60GGGGACCACTTTGTACAAGAAAGCTGGGTCCCTTTGA GTC SEQ ID NO: 61CACGACGTTGTAAAACGACG SEQ ID NO: 62 ATGTAATAGGAGTCACTATAGG

Platinum Taq DNA polymerase High Fidelity (Invitrogen Corporation;Carlsbad, Calif.) was used for PCR. The PCR conditions were as follows:

Components Volume Final Concentration dH₂O 35.5 μl 10 mM dNTP mixture(2.5 mM each) 4 μl 0.2 mM each 10 X High Fidelity PCR Buffer 5 μl 1X 50mM MgSO₄ 2 μl 2 mM Primer 1 (100 ng/μl) 1 μl Primer 2 (100 ng/μl) 1 μlTemplate (10 ng/μl) 1 μl Platinum Taq High Fidelity (5 U/μl) 0.5 μl μl

94° C.: 4 min (1 cycle)

94° C. 30 sec→55° C. 30 sec→68° C. 1 min (30 cycles)

68° C. 10 min (1 cycle)

4° C. (to completion)

The following conditions were used to purify PCR generated fragments:

Reagent: SNAP MiniPrep kit (Invitrogen Corporation, Carlsbad, Calif.).

Steps

(1) Mix 50 μl PCR product with 150 μl Binding Buffer. Mix well.

(2) Add 350 μl of Isopropanol. Mix well.

(3) Load the sample onto a SNAP MiniPrep Column.

(4) Centrifuge at 14000 rpm for 1 min. Discard the column flow through.

(5) Add 500 μl of Wash Buffer and centrifuge at 14000 rpm for 1 min.Discard the column flow through.

(6) Add 700 μl of 1× Final Wash Buffer and centrifuge at 14000 rpm for 1min. Discard the column flow through.

(7) Dry the column by centrifuge at 14000 rpm for 1 min.

(8) Transfer the column to a new centrifuge tube. Add 50 μl of dH₂O tothe column. Incubate at room temperature for 2-5 min. Centrifuge at14000 rpm for 1 min. Collect the flow through.

(9) DNA concentration measurement by UV absorbance at 260 nm.

TOPO Joining Reaction

For production of expression cassettes with secondary PCR, the followingjoining conditions were used: 33 5′ element (700 bp) 75 ng 3′ element(350 bp) 35 ng 500 mM Tris (pH7.5) 0.5 μl Topoisomerase (1 μg/μl) 0.5 μlCAT insert (700 bp) 150 ng dH2O enough to bring final volume to 5 μl

The reaction was performed at room temperature for 5-15 min. Half volumeof the reaction was used as template for the second round PCR withprimer set sequence #1 and sequence #4. PCR conditions were the same asabove except that the extension time was 2 min. After PCR, DNA waspurified as mentioned above. Purified DNA was used for transfection.

For production of expression cassette without secondary PCR, thefollowing joining conditions were used:

5′ element (700 bp) 510 ng 3′ element (350 bp) 230 ng 500 mM Tris (pH7.5) 1.5 μl Topoisomerase (1 μg/μl) 3 μl CAT insert (700 bp) 450 ng dH₂Oenough to bring final volume to 15 μl

The reaction was performed at 37° C. for 15 min. Proteinase K was addedto a final concentration of 50 μg/ml and the mixture was incubated at37° C. for 10 min. The treated DNA was ready for transfection.

Gene Expression Study

Three cell lines (suspension TRex-CHO, adherent TRex-CHO and adherentTRex-293 cell lines) were used as model cell lines to test theseexpression cassettes. Standard cell culture methods were used.Twenty-four well cell culture plates were used. Lipofectamine 2000 wasused as transfection reagent. Twenty-four hours after transfection,tetracycline was added at a final concentration of 1 μg/ml. For controlstudies, no tetracycline was added. Cells were incubated for another 24hours before lysis. Western blot was used for transfer of proteins andanti-V5 or anti-CAT antibody was used for detection.

Results and Discussion

The purpose of this study was to demonstrate that expression cassettescould be generated without secondary PCR. In this study, we compared theexpression data generated from an expression cassette produced using asecondary PCR step to that obtained using an expression cassetteproduced without a secondary PCR step. For the expression cassetteproduced with secondary PCR, about 1.2 μg/well of DNA was used fortransfection into 24-well plate format. For the expression cassettewithout secondary PCR, the product from one joining reaction was used(about 1.2 μg/well). The detection data showed that functionalexpression cassettes can be produced using the methods of the presentinvention, without using a secondary PCR step (FIG. 30).

Example 11 Generation of Gateway Compatible Cassettes with Topo ToolsMethods

Preparation of Adaptors

Equal amounts of sequence #7 and sequence #8 (see Table 8, above) weremixed in 40 mM NaCl and the mixture was denatured at 95° C. for 5 minand slowly cooled to room temperature to form the attB1 adaptor. Equalamounts of sequence #9 and sequence #10 (see Table 8, above) were mixedin 40 mM NaCl and the mixture was denatured at 95° C. for 5 min andslowly cooled to room temperature to form the attB2 adaptor.

TOPO Joining

CAT insert was generated as in example 10. The joining conditions wereas optimized above (see Examples 9 and 10): 35

attB1 adaptor (40 bp) 10 ng attB2 adaptor (40 bp) 10 ng 500 mM Tris (pH7.5) 0.5 μl Topoisomerase (1 μg/μl) 1 μl CAT insert (700 bp) 170 ng dH₂Osufficient to bring final volume to 5 μl

The reaction was performed at 37° C. for 15 min. SDS and EDTA were addedto a final concentration of 0.1% and 0.5 mM respectively. The mixturewas incubated at 37° C. for 15 min.

Purification

Water (15 Pμl) was added to the treated mixture. DNA was purified withSNAP MiniPrep kit (Invitrogen Corporation, Carlsbad, Calif.).

Steps

(1) Mix the treated product with 60 μl Binding Buffer. Mix well.

(2) Add 140 μl of Isopropanol. Mix well.

(3) Load the sample onto a SNAP MiniPrep Column.

(4) Centrifuge at 14000 rpm for 1 min. Discard the column flow through.

(5) Add 500 μl of Wash Buffer and centrifuge at 14000 rpm for 1 min.Discard the column flow through.

(6) Add 700 μl of 1× Final Wash Buffer and centrifuge at 14000 rpm for 1min. Discard the column flow through.

(7) Dry the column by centrifuge at 14000 rpm for 1 min.

(8) Transfer the column to a new centrifuge tube. Add 20 μl of dH₂O tothe column. Incubate at room temperature for 2-5 min. Centrifuge at14000 rpm for 1 min. Collect the flow through.

BP Reaction

BP reaction buffer 2 μl Purified product 5.5 μl pDONR 222 (300 ng/μl)0.5 μl BP clonase 2 μl

The reaction mixture was incubated at room temperature for 60 min then 1μl of Proteinase K (2 μg/μl) was added. The mixture was incubated at 37°C. for 15 min followed by 15 min at 75° C. to inactive the enzyme.

Transformation

The treated mixture was transformed into TOP10 competent cells(chemical) or electroporated into ElectroMax competent cells. Cells wereplated onto LP-Kanamycin plates and incubated at 37° C. overnight. Thenumber of colonies was counted. To make sure that insert was present inthese colonies, we designed primer sets (sequence #11 and #12) to docolony PCR. If insert was present, the PCR product would have produced aband of about 700 bp; if no insert was present, however, the PCR productband would be about 2.2 kb in size.

Results and Discussion

In this study, we wanted to demonstrate that PCR products produced withTOPO Tools sticky ends can be directly joined to attB1 and attB2adaptors. The joined product can be directly used in the BPrecombination reaction to create GATEWAY™ entry clones (Table 9).

TABLE 9 Colonies Generated from BP Reaction. Transformation TypeattB1-Cat-attB2 Vector only Chemical 188 0 Electroporation 8220 672

To further confirm the insert was present in these colonies, we picked18 positive colonies and 2 negative colonies to do PCR. PCR resultsshowed that right-sized product was present in all 18 colonies checked(FIG. 31).

The present invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

The following commonly owned, co-pending U.S. patent applications areincorporated herein by reference in their entireties: U.S. ProvisionalAppl. No. 60/254,510, filed Dec. 8, 2000; U.S. application Ser. No.09/732,914, filed Dec. 11, 2000; U.S. Provisional Appl. No. 60/291,972,filed May 21, 2001; U.S. Provisional Appl. No. 60/318,902, filed Sep.14, 2001; and U.S. Provisional Appl. No. 60/326,092, filed Sep. 28,2001.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

Example 12

In vitro transcription with the T7 bacteriophage promoter and RNApolymerase is commonly used to generate RNAs for downstream studies suchas probing of Northern blots, RNase protection assays, and RNAinterference. In order to produce the template molecule, the sequence ofinterest is usually cloned into a vector downstream of a T7 promotersequence or PCR amplified with primers including 20-30 nt T7 promotersat their 5′ ends. The first method requires subcloning, growth,isolation, and sometimes sequencing of the recombinant plasmid. Thesecond is rapid but requires 40-50 nt primers designed and synthesizedin advance of transcription.

T7 TOPO linkers provide a way to quickly and easily add a T7 promoter toan existing PCR product without the need to order new primers andwithout subcloning. A TOPO-charged linker containing the T7 promotersequence is joined to a Taq-generated PCR product in a 15 minutereaction. A secondary amplification with a linker-specific primer andone of the original gene-specific primers produces ample template for asmany T7 transcription reactions as needed and determines the orientationof the RNA that will be produced.

Here we test conditions for purification of a T7 TOPO linker, and weshow that it is capable of efficient ligation to actin and GFP PCRproducts and can direct T7 transcription from secondarily amplifiedtemplates at levels comparable to primary amplification products with aT7 promoter in one primer.

Materials and Methods

The following materials may be used to prepare T7 promoter linkers andattach them to a PCR product (e.g., the actin ORF), and then isolate aconstruct having the T7 promoter attached to the PCR product in thedesired orientation such that an RNA molecule can be transcribed with aT7 polymerase that corresponds to the sense or the antisense or bothstrands of the PCR fragment (see FIG. 42). T7 TOPO linkers are preparedas described below. A T7 secondary amplification primer having asequence that anneals to all or a portion of the T7 promoter such thatextension of the primer is in the direction of the attached PCR product.Various buffer, nucleotide and/or salt solutions may be employed in thereactions described, for example, salt solution (available fromInvitrogen Corporation, Carlsbad Calif., catalog number 46-0205), 10×PCRbuffer (available from Invitrogen Corporation, Carlsbad Calif., catalognumber 46-0121), 10 mM dNTPs (available from Invitrogen Corporation,Carlsbad Calif., catalog number 46-0344).

As an example of the use of the methods of the present invention, a PCRfragment containing all or portions of the actin gene is prepared usingforward and reverse actin control primers described below to amplify afragment from an actin control template. After attachment of the T7promoter to the fragment, in vitro transcription was performed using acommercially available T7 transcription kit.

Construction and Purification of a T7 Promoter Linker

The following oligos were synthesized and gel-purified:

T7topG (SEQ ID NO: 124)5′-pGACTCGTAATACGACTCACTATAGGGCCCTTATTCCGATAGTG-3′ T7botG (SEQ ID NO:125) pAGGGCCCTATAGTGAGTCGTATTACGAGTCAAAAAAAAAA- AA TOPO-5 (SEQ ID NO:126) pCAACACTATCGGAATA

A total of 50 μg oligos were annealed in a 1:1:3 molar ratio(T7topG:T7botG:TOPO-5) in 1×PNK buffer (New England Biolabs) and 200 mMNaCl by incubation in a thermal cycler for 5 min at 95° C., 5 min at 65°C., 5 min at 37° C., and 5 min at 25° C. 10 μg of annealed oligos werecharged in a 400 μl reaction consisting of 1×PNK buffer (New EnglandBiolabs) with 1 mM ATP, 20 U of polynucleotide kinase (New EnglandBiolabs), and 50 μg Vaccinia topoisomerase I for 15 min at 37° C. Thisresults in the attachment of the topoisomerase to the 3′ terminus of theT7 promoter linker that is to be attached to the PCR product.

The topoisomerase-charged promoter linker was purified using anAkta-FPLC and Unicom software ver. 4.00 (Amersham). 360 μl of thelinking reaction was loaded onto a 1 ml HiTrap SP Sepharose HPpre-packed column (Amersham) pre-equilibrated in buffer A (50 mMTris-HCl pH 7.0), washed at 0.5 ml/min with 11 ml buffer A (collected in1 ml fractions), and eluted with a 5 ml, 0-100% buffer B (50 mM Tris-HClpH 7.0, 1M NaCl) gradient followed by 3 ml of 100% buffer B. The eluatewas collected in 0.2 ml fractions. Representative chromatograms of theFPLC purification are shown in FIG. 43.

The peak fractions were identified by treating 4 μl of “load” fraction(unpurified linking reaction), 10 μl of each flow-through fraction, and16 μl of each eluate fraction with 5 μg proteinase K for 30 min prior toloading on a 10% polyacrylamide Novex TBE gel and electrophoresing for45 min at 200V. The gel was stained for 30 min in 0.2 μg/ml ethidiumbromide and destained by washing 10 min in ddH₂O. Linker concentrationcan be estimated by running low DNA mass ladder on the same gel andcomparing band intensities (see FIG. 44A).

The location of free topoisomerase among the fractions was determined byloading the same fraction volumes as above but without proteinase Ktreatment onto Novex 4-12% Tris-Bis NUPAGE gels. 0.3 μg of threetopoisomerase were run for comparison. The gels were electrophoresed for35 min at 200V in MES buffer and stained with Coomassie R-250 (see FIG.44B).

Three peak fractions (#33-35) of T7 TOPO linker were pooled, dilutedwith 2 volumes of storage buffer (60% glycerol, 67 μg/ml BSA, 50 mMTris-HCl pH 7.4, 0.3 mM EDTA, 1.3 mM DTT, 0.07% Triton-X 100), andstored at −20° C.

Plasmid Templates

pcDNA5/FRT/TO/GFP was from Invitrogen Corporation.

pBAD/TOPO-actin-as was created by TOPO cloning a blunt PCR productamplified with an actin forward, actinF, primer having the sequence5′-GCTCACCATGGATGATGATATCGC-3′ (SEQ ID NO:127) and an actin reverse,actinR, primer having the sequence 5′-GGAGGAGCAATGATCTTGATCTTC-3′ (SEQID NO:128) from the HeLa cDNA PCR control template (available fromInvitrogen Corporation, Carlsbad Calif., catalog number 46-0324) intopBAD/TOPO in the antisense orientation.

pUC 19/actin (FIG. 46A) was created by cloning of a BamHI-HindIIIdigested PCR product amplified from the HeLa cDNA template withBamHI-actinF primer having the sequence5′-CACGGATCCGCTCACCATGGATGATGATAT-CGC-3′ (SEQ ID NO:129) andactinR-HindIII primer having the sequence5′-CACAAGCTTGGAGGAGCAATGATCTTGATCTTC (SEQ ID NO:130) into BamHI-HindIIIdigested pUC19.

PCR

50 μl reactions were used for both primary and secondary amplificationsusing 10 pmol each primer, 0.2 mM dNTPs, 1×PCR buffer (from 10× stock,Invitrogen Corporation, Carlsbad Calif.), and 2.5 U Platinum Taq DNApolymerase or Recombinant Taq DNA polymerase. Primary reactions wereperformed using 1 ng of pBAD/TOPO-actin-as, pUC19/actin, orpcDNA5/FRT/TO/GFP plasmids as templates and actinF+actinR or GFPstart(5′-ATGGCTAGCAAAGGAGAAGAACTTT-3′ (SEQ ID NO:131))+GFPstop2(5′-TTATTTGTAGAGCTCATCCATGCCA-3′ (SEQ ID NO:132)) primers. Fortranscription control templates, the GFP and actin forward primers werepaired with reverse primers appended with a 5′ T7 promoter sequence(5′-GATGACTCGTAATACGACTCACTATAGGG-3′ (SEQ ID NO:133)). Secondaryreactions were the same as the primary reactions except 1 μl of T7 TOPOlinking reaction was used as template and either actinF or GFPstartprimers were combined with the linker-specific primer T7amp1(5′-GATGACTCGTAATACGACTCA-CTA-3′ (SEQ ID NO:134)).

GFP primary and secondary amplifications were incubated for 2 min at 94°C. followed by 30 cycles of 94° C. for 15 s, 57° C. for 30 s, and 72° C.for 45 s. Actin primary and secondary amplifications were incubated for2 min at 94° C. followed by 30 cycles of 94° C. for 15 s, 58° C. for 30s, and 72° C. for 1 min. All amplifications included a final extensionstep of 7 min at 72° C.

PCR product concentrations were estimated by running on 1.2% agarose-TAEgels with Low DNA Mass ladder and comparing band intensities.

T7 TOPO Linking Reactions

Linking was performed by combining 1 μl of pooled T7 TOPO linker eluatefractions in storage buffer with 1 μl of primary GFP or actin PCRreaction, 3 μl ddH₂O, and 1 μl salt solution (1.2M NaCl, 60 mM MgCl₂)and incubating at 37° C. for 15 min. Reactions were checked by running 8μl from a double reaction on a 6% polyacrylamide Novex TBE gel for 1 hrat 200V, staining in 0.2 μg/ml ethidium bromide, and destaining for 10min in ddH₂O. For the negative control, 1×TOPO storage buffer (2 vol.storage buffer, above, +1 vol. buffer A) was substituted for the pooledTOPO linker eluate in vitro transcription

1 μl-1.5 μl of secondary PCR reaction was mixed with 2 μl 75 mM NTPs(Amersham), 4.5 μl ddH₂O, 1 μl 10× transcription buffer (400 mM Tris-HClpH 8.0, 100 mM DTT, 20 mM spermidine, 100 mM MgCl₂), and 1.5 μl enzymemix (4 parts 50 U/μl T7 RNA polymerase, 1 part 40 U/μl RNaseOUT, 1 part0.6 U/μl yeast inorganic pyrophosphatase). Reactions were incubated for1 hr at 37° C. followed by addition of 0.51 μl DNase I (50 U/μl) andcontinued incubation for 15 min. 0.5 μl of each reaction was then mixedwith 4.5 μl ddH₂O and 5 μl of Gel Loading Buffer II (Ambion), denaturedfor 5 min at 95° C., cooled on ice for 5 min, and run on a 1.2%agarose-TAE gel for 45 min at 100V. The gel was stained for 30 min in 1μg/ml ethidium bromide and destained for 10 min in ddH₂O.

Results

T7 TOPO Linker Purification by FPLC

The T7 TOPO linker is produced by annealing three oligos, charging withVaccinia topoisomerase I, and purifying over SP Sepharose as describedin the Materials & Methods. The final product is a double-stranded oligocovalently bound to topoisomerase (FIG. 42A). The linker self-joins toTaq-generated PCR products in a 15 min reaction, forming a template forsecondary PCR and subsequent transcription (FIG. 42B).

FIG. 43A shows a chromatogram from the FPLC purification. The peak in UVabsorbance (254 nm) in flow-through fractions 2 and 3 corresponds tounbound linker, a small cleavage product, and ATP. The small peakcorresponding to TOPO-bound linker appears in fractions 30-33 and ismagnified in FIG. 43B. Gel electrophoresis (FIG. 44A) reveals thatfractions digested with proteinase K can be visualized as discrete bands(compare undigested load with load). The digested topoisomerase/linkercovalent complex runs at a lower molecular weight than annealed oligosalone due to cleavage of the oligo duplex by topoisomerase. Littlecovalent complex is evident in the flow-through fractions (F-T 2 through5). The lanes corresponding to elution fractions 29-40 demonstrate thatthe peak elution position of the linker is offset from the UV absorbancetrace on the chromatogram in FIG. 43A by approximately 3 fractions (0.6ml) to fractions 33-36.

Undigested fractions run on protein gels and stained with Coomassiereveal that free topoisomerase elutes in fractions 37-42 (FIG. 44B,compare to free topoisomerase control lane). T7 TOPO linker can bejoined to actin and GFP PCR products

T7 TOPO linker from peak fractions #33-35 was pooled and tested for theability to join with actin (pBAD/TOPO-actin-as template) and GFP(pcDNA5/FRT/TO/GFP template) test PCR products (see FIG. 45A andMaterials & Methods). A brief incubation with the linker causes aportion of each Platinum Taq-generated PCR product to shift into a moreslowly migrating band during agarose gel electrophoresis (FIG. 45B).This band presumably represents a single copy of the T7 linker joined toone end of the PCR product.

Secondary PCR reactions using the T7 linker specific primer T7amp1 andthe appropriate gene-specific forward primer (actinF or GFPstart)produce strong bands when the linker reaction is used as the templatebut not when control reactions lacking linker are used (FIG. 45C).Relatively weak bands are seen when only the T7amp1 primer is used inthe secondary amplification, possibly created from a small amount oftemplate carrying T7 linkers on both ends. Background bands are alsoevident in some negative control lanes, in which the mock linkingreactions (no T7 TOPO linker) were used as templates for the secondaryamplification.

T7 TOPO Linkers can be Used to Generate Competent Templates forTranscription

Approximately 40-50 ng of actin and GFP secondary PCR products (1.0 μl)(see FIG. 45C) or T7-actin (1.0 μl) and T7-GFP (1.5 μl) primary PCRproducts (see FIG. 45A) were used as templates in 10 μl transcriptionreactions as described in the Materials & Methods. The secondaryamplification products from both the actin and GFP linking reactions,but not from the corresponding negative controls, are competenttemplates for transcription by T7 RNA polymerase (FIG. 45D).

Transcription reactions using these secondary PCR reactions as templatesproduce equivalent or greater amounts of RNA to those using primary PCRreactions with the T7 promoter sequence added to the 5′ end of one ofthe primers, another common method of generating transcription templates(FIG. 45D). Thus the amplified product of the T7 TOPO linking reactionis a fully competent template. pUC19/actin can also function as an actintemplate for use with the T7 TOPO linker

Similar results were obtained using Recombinant Taq DNA Polymerase and apUC19/actin template (FIG. 46). Success with a non-Platinum polymeraseshows that use of the linkers does not require automatic hot start.

The amount of T7 TOPO linker in the peak fraction (#34) was estimated tobe 0.1 ng/μl in the final storage buffer. The linker concentration canbe roughly quantitated by running Low DNA Mass Ladder on a gel alongwith the proteinase K digested peak fractions as in FIG. 44A.

The success of PCR reactions, both primary and secondary, can beestimated by rough quantitation of the products on agarose gels as inFIG. 45A. 20-60 ng/μl should be acceptable for the primary reaction, asthe PCR products are in excess in the linking reactions. For thesecondary reaction, at least 25 ng/μl should be produced assignificantly lower levels will influence the yield of the transcriptionreaction.

Transcription reaction yields should be judged according to the criteriadeveloped for the forthcoming transcription kit.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. An isolated nucleic acid molecule comprising: (a)a first topoisomerase recognition site; (b) a first site specificrecombination site; (c) a nucleic acid segment; (d) a second sitespecific recombination site; and (e) a second topoisomerase recognitionsite; wherein the first site specific recombination site and the secondsite specific site do not recombine with each other.
 2. The nucleic acidmolecule of claim 1, wherein said nucleic acid molecule is a circularmolecule.
 3. The nucleic acid molecule of claim 1, wherein saidrecombination sites are selected from the group consisting of: (a) attBsites, (b) attP sites, (c) attL sites, (d) attR sites, (e) lox sites,(f) psi sites, (g) dif sites, (h) cer sites, (i) frt sites.
 4. Thenucleic acid molecule of claim 1, wherein said topoisomerase recognitionsite is recognized and bound by a type I topoisomerase.
 5. The nucleicacid molecule of claim 4, wherein said type I topoisomerase is a type IEtopoisomerase.
 6. A kit comprising the isolated nucleic acid molecule ofclaim
 1. 7. The nucleic acid molecule of claim 1, wherein the firsttopoisomerase recognition site and the first site specific recognitionsite are separated by from 0 to 100 nucleotides.